Caenorhabditis elegans is a well-established laboratory animal model and has been widely used in biological research. However, it is still a challenge to obtain a good amount of quality RNA from a limited number of C. elegans for gene expression studies. To address this issue, the present study has compared different conditions to preserve C. elegans for RNA extraction after the failure of an initial effort to use RNAlater-preserved worms for RNA extraction. The effects of different concentrations of proteinase K, different worm life stages, and different worm numbers on RNA extraction were also investigated. The best results were achieved under the following conditions: 1) adult worms that were either freshly prepared or quickly frozen in liquid nitrogen followed by storage at −80 °C; 2) disruption of C. elegans with proteinase K (1 mg/mL) in a lysis buffer (65 °C for 10 min) prior to extraction with Trizol agent. This method can provide a stable, rapid, and effective means to extract RNA from C. elegans with variable worm numbers from 20 to 200.•C. elegans for effective RNA extraction.RNAlater was inappropriate for preserving •C. elegans for RNA extraction.Proteinase K was verified for lysing a limited number of  Specification TableCaenorhabditis elegans, belonging taxonomically to the pseudocoelomates of Protostomia, has a simple body structure consisting of only about 1000 cells, and has approximately 20,000 protein-coding genes [C. elegans possess typical cell types of all metazoans such as muscle, nerve, intestine, and skin [C. elegans has been used extensively as a laboratory animal model for the research in neuroscience, reproduction toxicity, aging [y, aging ,4, and iy, aging .in situ, and allow the samples to be stored for a long period of time without jeopardizing the quality and quantity of RNA content [C. elegans has resilient cuticle that needs to be ruptured prior to RNA extraction [C. elegans using the reported method (data not shown). We also increased the number of RNAlater-preserved C. elegans and had no success in consistently obtaining a sufficient amount of quality RNA. This has led us to investigate if the use of RNAlater is appropriate to preserve C. elegans for extraction of RNA from the nematode, and subsequently to develop a rapid, effective, and stable method for extracting quality RNA from a limited number of C. elegans (from 20 to 200 worms). The results are reported herein.Transcription analysis can reveal the level of gene expression and lead to a better understanding of the physiological status of cells and/or animals. Efficient extraction of RNA to obtain a good amount and high quality of RNA remains critical in the success of transcription analysis. The traditional methods involve the use of phenol/chloroform or Trizol extraction . When th content . Since ttraction . Using ptraction , while ftraction .The resitraction . Recentltraction . HoweverC. elegans was cultured under standard laboratory condition [Escherichia coli OP50. The C. elegans was obtained from the Caenorhabditis Genetics Center (CGC), University of Minnesota, USA.ondition . Wild-tyC. elegans, worms were firstly collected into a 15 mL centrifuge tube using RNAse free water, then vortex and centrifuged at 300 rpm to remove the supernatant, repeated it three times to wash the worms. 20 washed worms were transferred in 5 μL RNase-free water to a new 200 μL RNase-free Eppendorf tube, mixed with 20 μL of basic lysis buffer containing 1 or 2 mg/mL of proteinase K , and then incubated at 65 °C for 2, 5, or 10 min followed by 85 °C for 1 min. The basic lysis buffer was consisted of 0.5% Triton (v/v), 0.5% Tween-20 (v/v), 0.25 mM EDTA, 2.5 mM Tris-HCl buffer (pH8.0), and 8% RNAsecure . Qubit RNA HS Assay kit  and Qubit® 2.0 Fluorometer (Invitrogen) were used to determine the level of RNA content in the worm lysates according to instructions from the manufacturer. Proteinase K at 1 mg/mL and incubation at 65 °C for 10 min were shown to be the best to fully lyse the worms. Therefore, these optimized conditions were subsequently used in the following experiments unless indicated otherwise.Adult worms collected from a NGM plate using RNase-free water and washed three times to remove bacteria were used in this experiment. To examine the effect of different proteinase K concentrations on the lysis of C. elegans. The storage conditions include: (I) fresh worms (no storage); (II) stored in RNAlater  at 4 °C for 24 h followed by storage at −80 °C until use; (III) stored in RNase-free water without RNAlater at 4 °C for 24 h followed by storage at −80 °C until use; (IV) frozen in liquid nitrogen and then stored at −80 °C until use. The methods to lyse worms and to determine the level of RNA content in worm lysates were the same as described above.Twenty adult worms (in 20 μL of RNAse-free water in a 2 mL Eppendof tube) stored under different conditions were used to determine total RNA content in the lysate of Twenty fresh adult worms or L4-stage worms were used to investigate the influence of growth stage over the yield of total RNA extract. After the lysis of worms to release RNA content using the optimized conditions described above, total RNA was extracted using Trizol™ Reagent according to instructions from the manufacturer . The yield of total RNA was determined by Nano-drop 1000 Spectrophotometer (Thermo Scientific).C. elegans and the yield of total RNA extracted by Trizol method were determined using the Qubit kit and fluorometer and Nano-drop 1000 Spectrophotometer, respectively.Scaled up experiments with different number of fresh worms were carried out to examine the efficiency of RNA extraction. Adult worms from 20 to 200 were lysed in the lysis buffer containing 1 mg/mL proteinase K based on the ratio of 1 μL lysis buffer per worm. After worm lysis using the optimized conditions, both the level of RNA content in the lysates of Total RNA extracted from adult worms was treated with TURBO DNA-free™ Kit  to remove DNA. One microgram of treated total RNA was used as the template for the synthesis of cDNA in a 20 μL reaction system using qScriptTM cDNA SuperMix  according to manufacturer’s protocol. One microliter cDNA template was used for quantitative PCR assays. The PCR assays were performed using a AB 7500 Real Time PCR System  with iTaq™ Universal SYBR Green Supermix  under the conditions as follows: 40 cycles of denaturation at 95 °C for 30 s, annealing at 56 °C for 1 min, extension at 72 °C for 30 s. The DNA sequences of PCR primers are listed in P ≤ 0.05 was accepted to be statistically significant.Results were expressed as mean ± S.D. Statistical significance of the data was performed by using one-way analysis of variance (ANOVA) in Statistical Product and Service Solutions , and a value of C. elegans samples were firstly used to test the efficiency of proteinase K in the lysis of worms and release of RNA content. Proteinase K could effectively disintegrate C. elegans to release RNA for extraction in within 10 min with 20 adult worms. However, when worms were not subjected to proteinase K treatment, no worm lysis and release of RNA content were observed. When the worms were incubated in the lysis buffer containing 1 mg/mL proteinase K, 5 or 10 min incubation appeared to be sufficient to release the total RNA content (P > 0.05), but much higher than 2 min incubation (P ≤ 0.05). However, 10 min incubation generated more consistent results in RNA extraction than 5 min. When the worms were treated with proteinase K at 1 mg/mL and 2 mg/mL, respectively, no dramatic difference in worm lysis (P > 0.05) was observed  was obtained from the worms preserved in RNAlater at 4 °C for 24h (Treatment II), which was similar to that of worms stored in RNase-free water without RNAlater at 4 °C for 24 h (Treatment III). The worms frozen by liquid nitrogen and transferred to −80 °C immediately for storage (Treatment IV) showed no significant difference in the level of released RNA content compared to the fresh worms (P > 0.05).As shown in C. elegans in RNAlater cannot effectively protect C. elegans RNA from degradation, even though the method has widely been used to preserve various types of cells and tissues for RNA extraction [C. elegans. The low efficiency of RNAlater in preserving C. elegans RNA could be attributed to the possibility that RNAlater cannot effectively penetrate the tough cuticle of the nematode to inhibit RNase in the body. Further studies are required to make a conclusion.All the results were reproducible and the RNAlater solution used for the experiment was proved functional properly when applied for extracting RNA from bacterial or Caco-2 cells. These data suggest that the storage of traction . To the As shown in versus frozen worm samples.The results of the scale-up experiments are shown in t values of three housekeeping genes from the 20-worm group, including snb-1, GAPDH, and RPL-4, and gene daf-16 that is a member in the insulin/insulin-like growth factor pathway of the nematode [The total RNA extract after removal of DNA was applied to real-time PCR assays to determine the quality of the RNA extract. The Cnematode , were 22C. elegans for effective RNA extraction. The method described in the present study that uses proteinase K to lyse the nematode offers an additional choice for rapid, simple, and consistent RNA extraction from a limited number of C. elegans.The commonly used method with RNAlater to preserve various types of cells and tissues for RNA extraction doesn’t appear to be appropriate for preserving None of the authors of this study has any financial interest or conflict with industries.


In essence, the procedure of biochemical separation yields three fractions: Input, eluate, and supernatent, which are subjected to RNA-seq. In this work, we present a bioinformatics workflow, which starts from RNA-seq data to infer gene-specific modification levels by a statistical model on a transcriptome-wide scale. Our workflow centers around the pulseR package, which was originally developed for the analysis of metabolic labeling experiments. We demonstrate how to analyze data without external normalization , given high efficiency of separation, and how, alternatively, scaling factors can be derived from unmodified spike-ins. Importantly, our workflow provides an estimate of uncertainty of modification levels in terms of confidence intervals for model parameters, such as gene expression and RNA modification levels. We also compare alternative model parametrizations, log-odds, or the proportion of the modified molecules and discuss the pros and cons of each representation. In summary, our workflow is a versatile approach to RNA modification level estimation, which is open to any read-count-based experimental approach.RNA modifications regulate the complex life of transcripts. An experimental approach called LAIC-seq was developed to characterize modification levels on a transcriptome-wide scale. In this method, the modified and unmodified molecules are separated using antibodies specific for a given RNA modification (e.g., m In particular, m6A was the first reversible RNA modification found in eukaryotes. It can be written by several methyltransferases and erased by two known demethylases (FTO and ALKBH5) [Until recently, mRNA modifications were underrepresented in the scientific literature. Specifically, internal modifications in mRNA and their roles in gene expression control were not considered in detail. The development of new sequencing techniques and discoveries from genetic studies have renewed the interest in understanding the effects of internal mRNA modifications . Over 15 ALKBH5) . Multipl ALKBH5) . The adv6A sites, which allowed the genome-wide mapping of m6A modifications [6A peaks in more than 7000 genes in human and mouse cells. These studies also showed that the methylation status of some m6A sites changes in response to stress. Global abundance level estimates in mRNA ranged from an m1A/A ratio of ≈0.02% [6A/A ratio of ≈0.4% [In 2012, two groups independently developed MeRIP-seq methods to assess the in vivo methylation state of mications ,5. Theseof ≈0.4% , but the6A-LAIC-seq approach [6A levels of particular genes or the relationship of m6A modification(s) to alternative RNA isoforms. To deconvolute the m6A epitranscriptome, mRNAs are not fragmented prior to RNA immunoprecipitation (IP), and full-length transcripts are sequenced in both m6A-positive and m6A-negative fractions post-IP.In this manuscript, we focus on the MeRIP-seq procedure, which allows to separate modified molecules from the pool of total RNA, provided that suitable antibodies are available. A variant thereof is the recently developed mapproach , which a6A-LAIC-seq dataset (NCBI accession SRP055176) consists of three fractions: The original pool, or input RNA, m6A modified RNA, or eluate, and m6A nonmodified RNA, referred to as supernatent. PolyA+ RNA was collected from two independent cell lines (GM12878 and H1-ESC) in 2 replicates and separated by antibody  into the aforementioned fractions. RNA-seq libraries were prepared as described in Reference [The published meference  and sequ6A IP and showed 100% efficiency in pulling down m6A-positive transcripts. In this case, for simplicity, we can assume that fraction cross-contamination is negligible, and we use the simpler model, where methylation level for a given gene is proportional to the mean expression  at the National Center for Biotechnology Information (NCBI). We removed adapter sequences and quality clipped reads using AR 3.0.3 . Ribosom2 v2.3.0 . All remAR 2.5.3 . Gene anE script . For all6A levels for each gene were quantified as in Molinie et al. using the fragment counts of the ERCC RNAs [6A levels were calculated as follows:E) and supernatent (S) counts (We refer to this approach as the LAIC-seq method. mRCC RNAs . A log–l) counts . The modThe pulseR package was developed specifically for metabolic labeling experiments, but can easily be adapted to epitranscriptomics workflow, where RNA modification levels are estimated from read count data. Intuitively, the RNA methylation level is represented as a proportion https://dieterich-lab.github.io/pulseR/articles/epitranscriptomics.html. We also provide the complete workflow in the supplementary files, including data and analysis scripts, as well as a full comparison between pulseR and the published estimates from Molinie et al. [In pulseR, samples are normalized for sequencing depth, and normalization between fractions is performed during the fitting procedure or else directly estimated from spike-in read counts. To simplify the notation, in the following sections, we do not include the normalization constants e et al. .6A level analysis in the absence of spike-ins.In the next section, we consider the case of mFor the analysis, we write the model in https://dieterich-lab.github.io/pulseR/). In summary, the model is fitted in several steps as follows:Fitting of gene specific parameters: Expression level fitting of normalisation factors (for a spike-ins free design): These are shared between the samples from the same fraction;fitting of global parameters: Size parameter for the negative binomial distribution (overdispersion in read counts).Additional information regarding counts, sample groupings, and how to specify boundaries on parameters and usage can be found in the pulseR documentation  was computed, assuming that the normalization factors and size parameter were fixed.In the pulseR package, the function If external spike-ins are available, we can use this information to determine the normalization factors, speeding up the fitting procedure. In summary, the input RNA is neglected, and spike-ins are used to directly relate eluate and supernatent fractions. This is possible because equal amounts of ERCC RNA spike-In mix were added to both fractions after IP. These counts are thus added to determine the scaling factors (without fitting). The formulas then reduce to:To use this information in pulseR, it suffices to pass the list of spike-ins as an additional argument, after adding the spike-ins to the count table with other genes. One of the fractions  must be assigned as a reference. As seen in We have presented a comprehensive workflow to assess the modification status of RNA molecules on a transcriptome-wide scale. We have demonstrated our approach based on a published LAIC-seq experiment, which involves an antibody-driven biochemical separation step on intact  RNA. Each input sample was separated into an eluate (modified) and supernatent (nonmodified) fraction. Target molecules of interest were turned into a table of read counts by an RNA sequencing experiment, which we had reanalyzed from raw data. Contrary to previous approaches, such as in Molinie et al. , we set We used a simple model to estimate the RNA methylation level, using a logit-transform in the absence of cross-contamination in two alternative scenarios: (a) Without spike-ins and (b) with ERCC spike-ins, to normalize read counts across fractions. In both scenarios, we showed that our estimates correlate very well with those obtained with the LAIC-seq method, and also with the data published in Molinie et al. , when coOur workflow is applicable to any current and future technique based on read counts from both fragmented and nonfragmented RNA, which can be separated into modified and unmodified fractions by biochemical means, chemical, or metabolic RNA labeling.References


The Montessori educational method has existed for over 100 years, but evaluations of its effectiveness are scarce. This review paper has three aims, namely to (1) identify some key elements of the method, (2) review existing evaluations of Montessori education, and (3) review studies that do not explicitly evaluate Montessori education but which evaluate the key elements identified in (1). The goal of the paper is therefore to provide a review of the evidence base for Montessori education, with the dual aspirations of stimulating future research and helping teachers to better understand whether and why Montessori education might be effective. She initially resisted going into teaching—one of the few professions available to women in the late 19th century—and instead became one of the very first women to qualify as a medical doctor in Italy. As a doctor she specialised in psychiatry and paediatrics. While working with children with intellectual disabilities she gained the important insight that in order to learn, they required not medical treatment but rather an appropriate pedagogy. In 1900, she was given the opportunity to begin developing her pedagogy when she was appointed director of an Orthophrenic school for developmentally disabled children in Rome. When her pupils did as well in their exams as typically developing pupils and praise was lavished upon her for this achievement, she did not lap up that praise; rather, she wondered what it was about the education system in Italy that was failing children without disabilities. What was holding them back and preventing them from reaching their potential? In 1907 she had the opportunity to start working with non-disabled children in a housing project located in a slum district of Rome. There, she set up her first 'Casa dei Bambini' ('children’s house') for 3–7-year olds. She continued to develop her distinctive pedagogy based on a scientific approach of experimentation and observation. On the basis of this work, she argued that children pass through sensitive periods for learning and several stages of development, and that children’s self-construction can be fostered through engaging with self-directed activities in a specially prepared environment. There was international interest in this new way of teaching, and there are now thousands of Montessori schools (predominantly for children aged 3–6 and 6–12) throughout the world.Central to Montessori’s method of education is the dynamic triad of child, teacher and environment. One of the teacher’s roles is to guide the child through what Montessori termed the 'prepared environment, i.e., a classroom and a way of learning that are designed to support the child’s intellectual, physical, emotional and social development through active exploration, choice and independent learning. One way of making sense of the Montessori method for the purposes of this review is to consider two of its important aspects: the learning materials, and the way in which the teacher and the design of the prepared environment promote children’s self-directed engagement with those materials. With respect to the learning materials, Montessori developed a set of manipulable objects designed to support children’s learning of sensorial concepts such as dimension, colour, shape and texture, and academic concepts of mathematics, literacy, science, geography and history. With respect to engagement, children learn by engaging hands-on with the materials most often individually, but also in pairs or small groups, during a 3-h 'work cycle' in which they are guided by the teacher to choose their own activities. They are given the freedom to choose what they work on, where they work, with whom they work, and for how long they work on any particular activity, all within the limits of the class rules. No competition is set up between children, and there is no system of extrinsic rewards or punishments. These two aspects—the learning materials themselves, and the nature of the learning—make Montessori classrooms look strikingly different to conventional classrooms.2 This is a very different goal to that of most education systems today, where the focus is on attainment in academic subjects such as literacy and mathematics. Thus when we ask the question, as this review paper does, whether children benefit more from a Montessori education than from a non-Montessori education, we need to bear in mind that the outcome measures used to capture effectiveness do not necessarily measure the things that Montessori deemed most important in education. Teachers and parents who choose the Montessori method may choose it for reasons that are not so amenable to evaluation.It should be noted that for Montessori the goal of education is to allow the child’s optimal development  to unfold.Despite its existence for over 100 years, peer-reviewed evaluations of Montessori education are few and they suffer from a number of methodological limitations, as will be discussed in Section 3. This review has three aims, namely to (1) identify some key elements of the Montessori educational method, (2) review existing evaluations of Montessori education, and (3) review studies that do not explicitly evaluate Montessori education but which evaluate the key elements identified in (1). My goal is to provide a review of the scientific evidence base for Montessori education, with the dual aspirations of stimulating future research and helping teachers to better understand whether and why Montessori education might be effective.6 Nevertheless, some elements of the method might still be beneficial, or could be successfully incorporated  into schools that do not want to carry the name 'Montessori' or to adhere fully to its principles. Pinpointing more precisely what—if anything—about the Montessori method is effective will enable a better understanding of why it works. Furthermore, it has been argued that there might be dangers in adopting wholesale and uncritically an educational method that originated over 100 years ago, in a world that was different in many ways to today’s.7 If the method is to be adopted piecemeal, which pieces should be adopted? As outlined previously, two important aspects of Montessori’s educational method are the learning materials, and the self-directed nature of children’s engagement with those materials. Some key elements of each of these aspects will now be considered in turn.The goal of this section is to isolate some key elements of the Montessori method, in order to better understand why, if Montessori education is effective, this might be, and what elements of it might usefully be evaluated by researchers. These are important considerations because there is considerable variability in how the Montessori method is implemented in different schools, and the name, which is not copyrighted, is frequently used without full adherence.The first learning materials that the child is likely to encounter in the Montessori classroom are those that make up the practical life curriculum. These are activities that involve pouring different materials, using utensils such as scissors, tongs and tweezers, cleaning and polishing, preparing snacks, laying the table and washing dishes, arranging flowers, gardening, doing up and undoing clothes fastenings, and so on. Their aims, in addition to developing the child’s skills for independent living, are to build up the child’s gross and fine motor control and eye-hand co-ordination, to introduce them to the cycle of selecting, initiating, completing and tidying up an activity (of which more in the next section), and to introduce the rules for functioning in the social setting of the classroom.3, the largest 10 cm3. In building the tower the child’s attention is being focused solely on the regular decrease in volume of successive cubes. There are no additional cues—different colours for example, or numbers written onto the faces of the cube—which might help the child to sequence the cubes accurately. Another piece of sensorial material, the sound boxes, contains six pairs of closed cylinders that vary in sound from soft to loud when shaken, and the task for the child is to find the matching pairs. Again, there is only one cue that the child can use to do this task: sound. The aim of the sensorial materials is not to bombard the child’s senses with stimuli; on the contrary, they are tools designed for enabling the child to classify and put names to the stimuli that he will encounter on an everyday basis.As the child settles into the cycle of work and shows the ability to focus on self-selected activities, the teacher will introduce the sensorial materials. The key feature of the sensorial materials is that each isolates just one concept for the child to focus on. The pink tower, for example, consists of ten cubes which differ only in their dimensions, the smallest being 1 cmThe sensorial materials, are, furthermore, designed as preparation for academic subjects. The long rods, which comprise ten red rods varying solely in length in 10 cm increments from 10 cm to 1 m, have an equivalent in the mathematics materials: the number rods, where the rods are divided into alternating 10 cm sections of red and blue so that they take on the numerical values 1–10. The touchboards, which consist of alternate strips of sandpaper and smooth paper for the child to feel, are preparation for the sandpaper globe in geography—a globe where the land masses are made of rough sandpaper but the oceans and seas are smooth. The touchboards are also preparation for the sandpaper letters in literacy and sandpaper numerals in mathematics, which the child learns to trace with his index and middle fingers.Key elements of the literacy curriculum include the introduction of writing before reading, the breaking down of the constituent skills of writing  before the child actually writes words on paper, and the use of phonics for teaching sound-letter correspondences. Grammar—parts of speech, morphology, sentence structure—are taught systematically through teacher and child-made materials.In the mathematics curriculum, quantities 0–10 and their symbols are introduced separately before being combined, and large quantities and symbols  and fractions are introduced soon after, all through concrete materials. Operations  are again introduced using concrete materials, which the child can choose to stop using when he is able to succeed without that concrete support.Principles running throughout the design of these learning materials are that the child learns through movement and gains a concrete foundation with the aim of preparing him for learning more abstract concepts. A further design principle is that each piece of learning material has a 'control of error' which alerts the child to any mistakes, thereby allowing self-correction with minimal teacher support.8 they do not, in isolation, constitute the Montessori method because they need to be engaged with in a particular way. Montessori observed that the young child is capable of concentrating for long periods of time on activities that capture his spontaneous interest.4 There are two features of the way that children engage with the learning materials that Montessori claimed promoted this concentration. The first is that there is a cycle of activity surrounding the use of each piece of material . If a child wishes to use the pink tower, for example, he will have to find a space on the floor large enough to unroll the mat that will delineate his work area, carry the ten cubes of the pink tower individually to the mat from where they are stored, then build the tower. Once he has built the tower he is free to repeat this activity as many times as he likes. Other children may come and watch, and if he wishes they can join in with him, but he will be able to continue on his own if he prefers and for as long as he likes. When he has had enough, he will dismantle the pink tower and reassemble it in its original location, ready for another child to use. This repeated and self-chosen engagement with the material, the lack of interruption, and the requirement to set up the material and put it away afterwards, are key elements aimed at developing the child’s concentration.10Important though the learning materials are,9). During those 3 h children are mostly free to select activities on their own and with others, and to find their own rhythm of activity, moving freely around the classroom as they do so. One might wonder what the role of the teacher is during this period. Although the children have a great deal of freedom in what they do, their freedom is not unlimited. The teacher’s role is to guide children who are finding it hard to select materials or who are disturbing others, to introduce new materials to children who are ready for a new challenge, and to conduct small-group lessons. Her decisions about what to teach are made on the basis of careful observations of the children. Although she might start the day with plans of what she will do during the work cycle, she will be led by her students and their needs, and there is no formal timetable. Hence the Montessori classroom is very different to the teacher-led conventional classroom with its highly structured day where short timeslots are devoted to each activity, the whole class is engaged in the same activities at the same time, and the teacher instructs at the front of the class.The second feature which aims to promote concentration is that these cycles of activity take place during a 3-h period of time  and the child-led manner of engagement with those materials  might potentially benefit development and learning over the teaching of the conventional classroom. We will return to many of the elements discussed here in the following two sections. ? Fourthly, there are presumably differences between Montessori schools (including the way in which the method is implemented) that might influence children’s outcomes, but studies rarely include more than one Montessori school, and sometimes not more than one Montessori class. Fifthly, and relatedly, there is the issue of 'treatment fidelity'—what counts as a Montessori classroom? Not all schools that call themselves 'Montessori' adhere strictly to Montessori principles, have trained Montessori teachers, or are accredited by a professional organisation. A sixth, and again related, point is that children’s experiences in Montessori education will vary in terms of the length of time they spend in Montessori education, and the age at which they attend. Finally, the numbers of children participating in studies are usually small and quite narrow in terms of their demographics, making generalisation of any results problematic. These methodological issues are not limited to evaluations of Montessori education, of course—they are relevant to much of educational research.12 Parents choose their child’s school for a host of different reasons,13 and randomisation is important in the context of Montessori education because parents who choose a non-conventional school for their child might be different in relevant ways from parents who do not, for example in their views on child-rearing and aspirations for their child’s future. This means that if a study finds a benefit for Montessori education over conventional education this might reflect a parent effect rather than a school effect. Furthermore, randomisation also controls for socio-economic status (SES). Montessori schools are often fee-paying, which means that pupils are likely to come from higher SES families; children from higher SES families are likely to do better in a variety of educational contexts.16 A recent report found that even public  Montessori schools in the USA are not representative of the racial and socioeconomic diversity of the neighbourhoods they serve.17 However, random assignment of children to Montessori versus non-Montessori schools for the purposes of a randomised control trial would be very difficult to achieve because it would take away parental choice.Of these, the lack of randomised control trials is particularly notable given the recognition of their importance in education.18 They compared children in Montessori and non-Montessori education and from two age groups—5 and 12-year olds—on a range of cognitive, academic, social and behavioural measures. Careful thought was given to how to overcome the lack of random assignment to the Montessori and non-Montessori groups. The authors’ solution was to design their study around the school lottery that was already in place in that particular school district. All children had entered the Montessori school lottery; those who were accepted were assigned to the Montessori group, and those who were not accepted were assigned to the comparison (other education systems) group. Post-hoc comparisons showed similar income levels in both sets of families. Although group differences were not found for all outcome measures, where they were found they favoured the Montessori group. For 5-year olds, significant group differences were found for certain academic skills , a measure of executive function (the card sort task), social skills  and theory of mind . For 12-year olds, significant group differences were found on measures of story writing and social skills. Furthermore, in a questionnaire that asked about how they felt about school, responses of children in the Montessori group indicated that they felt a greater sense of community. The authors concluded that 'at least when strictly implemented, Montessori education fosters social and academic skills that are equal or superior to those fostered by a pool of other types of schools'.18Arguably the most robust evaluation of the Montessori method to date is that by Lillard and Else-Quest.19 but Lillard and Else-Quest’s response is that the school was faithful to Montessori principles, which suggests that the results might be generalisable to other such schools.20 That fidelity might impact outcomes has long been of concern,21 and was demonstrated empirically in a further, longitudinal, study,6 that compared high fidelity Montessori classes , 'supplemented' Montessori classes , and conventional classrooms. Children in these classes were 3–6 years old, and they were tested at two time-points: towards the beginning and towards the end of the school year. Although the study lacked random assignment of children to groups, the groups were matched with respect to key parent variables such as parental education. As in Lillard and Else-Quest’s earlier study,18 outcome measures tapped a range of social and academic skills related to school readiness . There were two research questions: firstly, do preschool children’s school readiness skills change during the academic year as a function of school type, and secondly, within Montessori schools, does the percentage of children using Montessori materials in a classroom predict children’s school readiness skills at the end of the academic year? Overall, the answer to both questions was “yes”. Children in the high-fidelity Montessori school, as compared with children in the other two types of school, showed significantly greater gains on measures of executive function, reading, math, vocabulary, and social problem-solving. Furthermore, the degree to which children were engaged with Montessori materials significantly predicted gains in executive function, reading and vocabulary. In other words, treatment fidelity mattered: children gained fewer benefits from being in a Montessori school when they were engaged in non-Montessori activities.Their study has been criticised for using just one Montessori school,6 In a move to explore the role of the Montessori materials further, a more recent experimental study22 removed supplementary materials, to leave just the Montessori materials, from two of the three classrooms in a Montessori school that served 3–6-year olds. Over a period of 4 months children in the classrooms from which supplementary materials were removed made significantly greater gains than children from the unchanged classroom on tests of letter-word identification and executive function, although not on measures of vocabulary, theory of mind, maths, or social problem-solving. The authors acknowledge weaknesses in the study design, including the small number of participants (just 52 across the three classrooms) and the short duration. Nevertheless, the study does provide a template for how future experimental manipulations of fidelity to the Montessori method could be carried out.This study does not demonstrate definitively that the Montessori materials drove the effect: there might have been other differences between the high and lower fidelity classrooms—such as the teachers’ interactions with their pupils—that were responsible for the difference in child outcomes.6 For example, two early randomised control trials to evaluate Head Start in the USA did not find any immediate benefit of Montessori preschool programmes over other types of preschool programmes.24 In both programmes, only 4-year olds were included, whereas the ideal in Montessori preschool programmes is for 3–6 year olds to be taught in the same class in order to foster child-to-child tutoring.6 Furthermore, in one of the programmes23 the ideal 3-h work cycle was reduced to just 30 min.6 A more recent study of older children compared 8th grade Montessori and non-Montessori students matched for gender, ethnicity and socio-economic status.25 The study found lower scores for Montessori students for English/Language Arts and no difference for maths scores, but the participating Montessori school altered the “ideal” by issuing evaluative grades to pupils and introducing non-Montessori activities.6Fidelity is important because variation in how faithful Montessori schools are to the 'ideal' is likely to be an important factor in explaining why such mixed findings have been found in evaluations of the Montessori method.23 social and cognitive benefits did emerge for children who had previously attended Montessori preschools and then moved to conventional schools, but these benefits did not emerge until adolescence, while a follow-up study26 found cognitive benefits in Montessori males only, again in adolescence. Although such 'sleeper effects' have been widely reported in evaluations of early years interventions, they may be artefacts of simple measurement error and random fluctuations.27 Importantly, if the argument is that lack of fidelity to the Montessori method is responsible for studies not finding significant benefits of Montessori education at younger ages, it is not logical to then credit the Montessori method with any benefits that emerge in follow-up studies.These same limitations then make it difficult to interpret studies that have found 'later' benefits for children who have been followed up after a subsequent period of conventional education. In one of the studies discussed earlier,28 Compared to the non-Montessori group , the Montessori group scored significantly higher on maths and science, but no differences were found for English and social studies. What might explain this differential effect? The authors suggested that the advantages for maths might be driven by the materials themselves, compared to how maths is taught in conventional classes.28 Alternatively, or perhaps in addition, children in Montessori classrooms might spend more time engaged in maths and science activities compared to children in conventional classes, with the amount of time spent on English and social studies not differing. However, the authors were unable, within the design of their study, to provide details of exactly how much time children in the Montessori school had spent doing maths, science, English and social studies, in comparison to the time that children in conventional classes were spending on those subjects.Some studies report positive outcomes for certain curricular areas but not others. One, for example, investigated scores on maths, science, English and social studies tests in the final years of compulsory education, several years after children had left their Montessori classrooms.29 speculated that Montessori would have found much to appreciate in one of the non-Montessori comparison classes, including its 'freedom for the children ; its planned environment ; its non-punitive character ; and its emphasis on concentration (the children sustained activity without direct supervision for relatively long periods of time)'. In some evaluations, the differences between Montessori and conventional classrooms might not actually be so great, which might explain why benefits of being educated in a Montessori classroom are not found. And even if the Montessori approach to teaching a particular curriculum area is different to those used in conventional classrooms, there are likely to be different, equally-effective approaches to teaching the same concepts. This is a suggested explanation for the finding that although children in Montessori kindergartens had an advantage relative to their conventionally-educated peers for base-10 understanding in mathematics, they did not maintain this advantage when tested 2 years later.30Just as knowing what is going on in the Montessori classroom is vital to being able to interpret the findings of evaluations, so is knowing what is going on in the comparison classrooms. One of the earliest evaluations of Montessori education in the USA31 compared just 14 four and five-year-old children who attended a Montessori nursery school with 14 four and five-year olds who attended a conventional nursery school . In a non-verbal creativity task, involving picture construction, they were given a blank sheet of paper, a piece of red gummed paper in the shape of a curved jellybean, and a pencil. They were then asked to think of and draw a picture in which the red paper would form an integral part. Each child’s construction was rated for originality, elaboration, activity, and title adequacy, and these ratings were then combined into a 'creativity' score. The group of conventionally-schooled children scored almost twice as high as the Montessori group. A second task involved the child giving verbal descriptions of seven objects: a red rubber ball, a green wooden cube, a short length of rope, a steel mirror, a piece of rectangular clear plastic, a piece of chalk, and a short length of plastic tubing. Each description was scored as to whether it was functional  or whether it was a description of the object’s physical characteristics . Like the non-verbal creativity task, this task differentiated the two groups: whereas the conventionally educated children gave more functional descriptions , the Montessori children gave more physical descriptions . A third task, the Embedded Figure Test, involved the child first being presented with a stimulus figure and then locating a similar figure located in an embedding context. Both accuracy and speed were measured. While the two groups did not differ in the number of embedded figures accurately located, the Montessori group completed the task significantly more quickly. The fourth and final task required children to draw a picture of anything they wanted to. Drawings were coded for the presence or absence of geometric figures and people. The Montessori group produced more geometric figures, but fewer people, than the conventional group.While most evaluations are interested in traditional academic outcomes or factors related to academic success such as executive functions, a small number have investigated creativity. For example, an old study31 They wrote that 'The study does, however, support the notion that differing preschool educational environments yield different outcomes' and 'Montessori children responded to the emphasis in their programme upon the physical world and upon a definition of school as a place of work; the Nursery School children responded on their part to the social emphasis and the opportunity for spontaneous expression of feeling'. They did not, however, compare and contrast the particular features of the two educational settings that might have given rise to these differences.The authors were careful not to cast judgement on the performance differences between the two groups.32 Seven to twelve-year olds were tested longitudinally on five tasks tapping different aspects of creativity. 'Divergent' thinking tasks required children to (1) think of unusual uses for a cardboard box, (2) come up with ideas for making a plain toy elephant more entertaining, and (3) make as many drawings as possible starting from pairs of parallel lines. 'Integrative' thinking tasks required children to (1) invent a story based on a title that was provided to them, and (2) invent a drawing incorporating six particular shapes. Their sample was bigger than that of the previous study,31 comprising 40 pupils from a Montessori school and 119 from two conventional schools, and pupils were tested in two consecutive years (no information is provided about whether pupils from different schools were matched on any variable other than age). For both types of task and at both time-points the Montessori-educated children scored higher than the conventionally-educated children. Again, the authors made little attempt to pinpoint the precise differences between schools that might have caused such differences in performance.Creativity has been studied more recently in France.33 demonstrated that the practical life materials can be efficacious in non-Montessori classrooms. More than 50 different practical life exercises were introduced into eight conventional kindergarten classes, while five conventional kindergarten classes were not given these materials and acted as a comparison group. The outcome measure was a fine motor control task, the 'penny posting test', whereby the number of pennies that a child could pick up and post through a one-inch slot in a can in two 30 s trials was counted. At pre-test the treatment and comparison groups did not differ in the number of pennies posted, but at post-test 6 months later the treatment group achieved a higher score than the comparison group, indicating finer motor control. A nice feature of this study is that teachers reported children in both groups spending the same amount of time on tasks designed to support fine motor control development, suggesting that there was something specific to the design of the practical life materials that was more effective in this regard than the conventional kindergarten materials on offer. And because the preschools that had used the practical life activities had introduced no other elements of the Montessori method, the effect could be confidently attributed to the practical life materials themselves.None of the studies discussed so far has attempted to isolate individual elements of the Montessori method that might be accounting for any of the positive effects that they find. There are several studies, however, that have focused on the practical life materials. A quasi-experimental study34 investigated the potential benefits of the practical life materials for fine motor control by comparing 5-year olds in Montessori kindergarten programmes with 5-year olds in a conventional programme (reported to have similarities in teaching mission and pupil background characteristics) on the 'flag posting test'. In this task, the child was given a solid hardwood tray covered with clay in which there were 12 pinholes. There were also 12 paper flags mounted on pins, six to the right of the tray and six to the left, and the child’s task was to place the flags one at time in the holes. The child received three scores: one for the amount of time taken to finish the activity, one for the number of attempts it took the child to put each flag into the hole, and one for hand dominance (to receive a score of 1 (established dominance) the child had to consistently use the same hand to place all 12 flags, whereas mixed dominance received a score of 0). Children were pre-tested at the beginning of the school year and post-tested 8 months later. Despite the lack of random assignment to groups, the two groups did not differ on pre-test scores, but they did at post-test: at post-test the Montessori group were significantly faster and significantly more accurate at the task, and had more established hand dominance. However, no attempt was made to measure how frequently children in both groups engaged with materials and activities that were designed to support fine motor control development. Furthermore, the children in the Montessori classrooms were at the age where they should also have been using the sensorial materials, some of which  are manipulated by holding small knobs, and whose use could potentially enhance fine motor control. At that age children would also have been using the 'insets for design', materials from the early literacy curriculum designed to enhance pencil control. Therefore, although the results of this study are consistent with the practical life materials enhancing fine motor control, the study does not securely establish that they do.An extension of this study35 introduced practical life exercises into conventional kindergarten classes, while control kindergarten classes were not given these materials. 15 min were set aside in the experimental schools’ timetable for using the practical life materials, and they were also available during free choice periods. This time the outcome measure at pre-test and post-test was not fine motor skill but attention. There were benefits to attention of being in the experimental group, but only for girls—boys showed no such benefits. The differential gender impact of the practical life materials on the development of attention is puzzling. Girls did not appear to engage with the materials more than boys during the time that was set aside for using them, but no measure was taken of whether girls chose them more frequently than boys during the free choice periods. Similarly, there were no measurements of the time that children in both the experimental and control groups spent engaged in other activities that might have enhanced fine motor control. Nor is it clear whether it was the fine motor practice directly or rather the opportunity to select interesting activities  that was responsible for the benefits to attention that were recorded for girls.A further study36 The authors did not establish exactly which elements of the Montessori method might be responsible for this finding, but they did speculate that the following might be relevant: “students were provided at least 2 h per day to exercise choice and self-regulation; none of the students received mandatory grades; student grouping was primarily based on shared interests, not standardised tests; and students collaborated often with other students”. The authors did not evaluate the Montessori and non-Montessori groups on any measures of academic outcomes, but given the links between academic success and motivation at all stages of education (they provide a useful review of this literature), this link would be worth investigating in Montessori schools.Finally, it has been found that young adolescents in Montessori middle schools show greater intrinsic motivation than their peers in conventional middle schools .This section has discussed studies that have evaluated the Montessori method directly. To date there have been very few methodologically robust evaluations. Many suffer from limitations that make it challenging to interpret their findings, whether those findings are favourable, neutral or unfavourable towards the Montessori method. However, while randomised control trials could (and should) be designed to evaluate individual elements of the Montessori method, it is difficult to see how the random assignment of pupils to schools could work in practice  and decode them (in reading). One of the criticisms of synthetic phonics is that it teaches letters and sounds removed from their meaningful language context, in a way that analytic phonics does not.44 It has long been recognised that the goal of reading is comprehension. Reading for meaning requires both code-based skills and language skills such as vocabulary, morphology, syntax and inferencing skills,45 and these two sets of skills are not rigidly separated, but rather interact at multiple levels.46 Indeed, phonics instruction works best where it is integrated with text-level reading instruction.40 The explicit teaching of phonics within a rich language context—both spoken and written—is central to the Montessori curriculum. No evaluations have yet pitted phonics teaching in the Montessori classroom versus phonics teaching in the conventional classroom, however, and so whether the former is differentially effective is not known.The Montessori approach to teaching phonics is certainly systematic. Many schools in the UK, for example, use word lists drawn from Morris’s 'Phonics 44'.48 Proficiency in these skills predicts the quality of children’s written compositions.50 In the Montessori classroom these skills are worked on independently before being brought together, but they can continue to be practised independently. A growing body of research from conventional and special education classrooms demonstrates that the specific teaching of the component skills of writing improves the quality of children’s written compositions.54Research into writing supports Montessori’s view that writing involves a multitude of component skills, including handwriting, spelling, vocabulary and sentence construction.55 Some of the recommended activities, such as 'help children to recognise, name, and compare shapes, and then teach them to combine and separate shapes'55 map exactly on to Montessori’s sensorial materials such as the geometric cabinet and the constructive triangles. Other activities such as 'encourage children to label collections with number words and numerals'55 map onto Montessori’s early mathematics material such as the number rods, the spindle box and the cards and counters. The importance of conceptual knowledge as the foundation for children being able to understand fractions has been stressed.56 The Montessori fraction circles—which provide a sensorial experience with the fractions from one whole to ten tenths—provide just such a foundation, as do practical life exercises such as preparing snacks (how should a banana be cut so that it can be shared between three children?) and folding napkins.With respect to teaching mathematics to young children, there are many recommendations that Montessori teachers would recognise in their own classrooms, such as teaching geometry, number and operations using a developmental progression, and using progress monitoring to ensure that mathematics instruction builds on what each child knows.4 These are important parts of the complex cognitive construct of executive functions (EFs), which also include inhibition, working memory and planning. Put simply, EFs are the set of processes that allow us to control our thoughts and actions in order to engage in motivated, goal-directed behaviour. That EFs are critical for academic success is backed by a wealth of research evidence.61 Given this key role, EFs have become the target for a number of individually-administered interventions, full curricula, and add-ons to classroom curricula, such as CogMed , Tools of the Mind,62 PATHS , music, yoga and martial arts. A review study compared these, including Montessori education, and concluded that compared to interventions such as CogMed that solely target EFs, 'school curricula hold the greatest promise for accessibility to all and intervening early enough to get children on a positive trajectory from the start and affecting EFs most broadly'.63Finally in this section, it is worth returning to the sustained attention and self-regulation that have been argued to characterise children’s engagement with the learning materials in the Montessori classroom.6 However, by its very nature, of course, greater adaptability means lower fidelity. This paper has discussed evidence that children may benefit cognitively and socially from Montessori education that is faithful to its creator’s principles, but it is less clear that adapted forms—which usually result in children spending less time engaged with self-chosen learning materials—are as effective. Nevertheless, studies suggest that the practical life materials can be usefully introduced into non-Montessori classrooms to support the development of young children’s fine motor skills and attention, and there is ample evidence from the wider educational literature that certain elements of the Montessori method—such as teaching early literacy through a phonic approach embedded in a rich language context, and providing a sensorial foundation for mathematics education—are effective. It has not been possible in this paper to give an exhaustive discussion of all the elements of Montessori education that might be beneficial, for example the lack of extrinsic rewards, the reduced emphasis on academic testing and lack of competition between pupils, the 3-year age-banding that fosters cross-age tutoring, or the presence of a trained teacher in the early years classroom.Montessori education has been in existence for over a hundred years. Such longevity could well be due, at least in part, to its adaptability.64 Yet Montessori teachers often feel fear or uncertainty about being able to apply Montessori’s theories in new and innovative ways while still adhering to her underlying philosophical principles.65 Ultimately, only empirical research, undertaken by teachers and researchers working together, can be our guide, because the questions that need answering are empirical in nature. Neuroscientific research—using neuroimaging methods which were not available in Montessori’s day—might also play a guiding role. For example, Montessori was prescient in her views that adolescence was a special time in development where the individual required a specially-designed form of education to address their needs.66 Recent neuroimaging evidence points to adolescence as indeed being an important period for neural development, particularly for areas involved in executive functions and social cognition.68 Montessori did not fully develop her ideas for the education of 12–18-year olds during her lifetime, but it is an area where current Montessorians might be able to take over the reins. Although some Montessori schools take pupils up to the age of 18, they are few and far between, and to my knowledge there are no published evaluations of their effectiveness. Developing a Montessori education for this age group in conjunction with the best of our current knowledge of developmental cognitive neuroscience has the potential to make a very positive contribution.Where does this leave Montessori education more than 100 years after its birth, and more than 60 years after the death of its creator? As others have noted, Montessori was a scientist who truly valued the scientific method and would not have expected her educational method to remain static.69 it is interesting to note that the Montessori method is now being adapted for use with dementia patients, with the aim of improving functioning in activities of daily living, such as feeding, and in cognition. There is strong evidence for a reduction in difficulties with eating, weak evidence for benefits on cognition, and mixed evidence for benefits on constructive engagement and positive affect.70 However, the quality of studies varies across domains; those evaluating effects on cognition have been of rather poor quality so far, and they have not yet examined whether there might be long-term effects. Nevertheless, given the challenges to developing successful medication for patients with Alzheimer’s disease despite a detailed knowledge of changes in their neurobiology, it would be sensible to continue the search for successful behavioural interventions alongside that for medical interventions.71 One method for delivering Montessori-based activities to the elderly is via inter-generational programmes, whereby older adults with dementia are supported in teaching Montessori-based lessons to preschool children. Benefits have been reported for the adults involved,72 but whether the children also benefit in particular ways from such inter-generational teaching has not been evaluated. Nor is it known whether a Montessori education in childhood or Montessori-based activities experienced in later life can protect the executive control circuits of the brain, as has been proposed for bilingualism.73 A lifespan approach to the evaluation of the Montessori method involving both behavioural and neuroimaging methods might be valuable.Nor did Montessori consider using her method with the elderly. In the context of a rapidly aging population and increasing numbers of elderly adults with acquired cognitive impairments such as those that result from Alzheimer’s disease,74 and towards and away from children working individually.75 This means that elements of the Montessori method will sometimes be in vogue and sometimes not. It is therefore particularly important that Montessori teachers understand the evidence base that supports, or does not support, their pedagogy.In sum, there are many methodological challenges to carrying out good quality educational research, including good quality research on the Montessori method. Arguably the most obvious challenge to emerge from the literature reviewed here is the practical difficulty of randomly allocating pupils to Montessori and non-Montessori schools in order to compare outcomes. The majority of studies have relied instead on trying to match pupils and teachers in Montessori and non-Montessori schools on a number of different variables, with the concomitant danger that unidentified factors have contributed to any difference in outcomes. Even if randomisation is achievable, studies need to be conducted on a large enough scale to not only allow generalisations to be made beyond the particular schools studied, but to also allow investigation of which children the Montessori method suits best. On a more optimistic note, recent experimental studies—whereby features of existing Montessori classrooms are manipulated in some way, or features of the Montessori method are added to non-Montessori classrooms—hold promise for investigating the effectiveness of particular elements of the Montessori method. The evidence base can be strengthened yet further by drawing on research of educational interventions with which it shares certain elements, and by drawing on related research in the science of learning. National and regional education systems are beset by regular swings of the pendulum, for example towards and away from phonics,


Toxoplasma gondii and Trichinella sp. T6 (parasitic infectious agents), Babesia microti and Borrelia burgdorferi (tick-vector borne agents), and Treponema denticola and Porphyromonas gingivalis, agents causing chronic gingivitis. Primary aligned reads of each agent in each tissue sample were quantitated with SAMtools. We found small percentages (<0.1%) of transcriptomes aligned with B. microti, B. burgdorferi, T. denticola, and P. gingivalis genomes and larger percentages aligned with T. gondii (0.1–0.2%) and Trichinella sp. T6 (1.0–1.1%) genomes. In AD specimens, but in no others, primary aligned transcriptome percentages, although small, approached significance for being greater in AD compared to CTL samples for B. burgdorferi (p = 0.067) and P. gingivalis (p = 0.068). Genes’ expressions in postmortem tissues of AD and ALS but not PD revealed significant changes among disease-associated microglial (DAM) genes. Infectious agents’ transcripts can be detected in RNA seq reads of both NDD and CTL tissues and vary from agent to agent. Expressions of Stage 1 and Stage 2 DAM genes significantly changed, suggesting the presence of Stages 1 and 2 DAM in our NDD tissue samples.Nervous tissues from both humans with neurodegenerative diseases (NDD) and animals with genetic models of human NDD, such as rare monogenic causes of Amyotrophic Lateral Sclerosis (ALS), Alzheimer’s disease (AD), and Parkinson’s disease (PD), show activated microglia, suggesting a potential causal role for inflammation in pathogenesis of NDD. We performed paired-end (PE) RNA sequencing (RNA seq) of total RNA’s extracted from frozen sections of cervical spinal cords from ALS and CTL subjects, frontal cortical gray matter ribbons of AD and CTL subjects, and ventral midbrains of PD and CTL subjects. Trimmed PE reads were aligned against the hg38 human transcriptome using Tophat2/Bowtie2 (ALS) or HISAT2 (AD and PD) and quantitated with Cufflinks. PE reads were also aligned using Bowtie2 against genomes from representative species of  Microglia are CNS-resident immune cells that can serve both beneficial (reduction of immune responses) and detrimental (activation of neurotoxic immune responses) functions  .op cit above).Because activated microglia can produce known neurotoxic substances, such as tumor necrosis factor alpha (TNF-α) , microglBy sorting brain immune cells and carrying out massively parallel RNA sequencing (RNA seq) on these cells over the course of disease progression in the 5X FAD mouse model of human AD Such DAM appeared to originate from “homeostatic” microglia .We acquired postmortem samples of CNS tissues from sporadic NDD cases and carried out moderate-high density PE RNA sequencing on total RNA to seek systems biology understandings of disease pathogenesis in ALS, AD, and PD   to determine if any of the PE RNA seq reads aligned with bacteria or parasite genomes downloaded from the NIH genome site. We then assayed the expression in each NDD tissue sample (of CTL) of genes associated with homeostatic microglia, Stage 1 or Stage 2 DAM as defined by -6 (B. microti) and ∼1.1 (Trichinella sp. T6). These results are summarized in Borrelia burgdorferi and Porphyromonas gingivalis that approached significance . In no other pair did we observe a situation where NDD >CTL for transcript abundance of infectious disease agents. Because all of our total RNA extracts were treated with DNAase and used cDNA’s generated for multiplex RNA seq reads, we are confident that alignments represent NDD tissue transcripts  aligned to infectious agent genomes . We did note a substantial difference in abundance of transcripts aligned to the genome of Trichinella sp. T6 compared to all others examined.In our RNA seq studies we obtained between ∼56 and ∼172 million PE reads . From these PE reads, we found wide variation in the number of reads primarily aligned with infectious agents’ genomes. When expressed as % of total PE reads, we observed between ∼3.2 × 10In the second part of our study, we examined expressions of genes associated with homeostatic microglia and Stage 1 and Stage 2 disease-associated microglia (DAM), as defined by Two-way ANOVA tests showed significant variation by gene for the ALS  and AD  samples but not for the PD samples. Individual unpaired t-tests revealed significant changes for all gene groups in the ALS and two gene groups in the AD, but none for the PD samples. We note that for the Stage 1 DAM analysis in the AD samples, we found a significant decrease in expression of P2RY12 (purinergic receptor P2Y12), also observed by Toxoplasma and Trichinella (By querying ∼52 to ∼172 million RNA seq paired-end (PE) reads in tissues from the three major adult NDD’s, we found that highly variable numbers of primary alignments could be found for several infectious agents known to affect humans. This is a limited list of infectious agents, and we appreciate that others could have been selected. These agents were selected based on documented infections in humans and availability of genomes. CNS involvement with parasitic infections, in particular, is known for chinella .B. burgdorferi, P. gingivalis, and Treponema denticola) and much higher alignment frequencies (for Trichinella sp. T6). With one exception (AD), we found no evidence that alignment frequencies in a NDD tissue set was greater than in CTL tissues from the same brain region. These findings suggest it is unlikely that sub-clinical infections could account for inflammation associated with NDD tissues we examined with RNA-seq. This assessment is tempered by the possibility that RNA species from the infectious agents could have been produced earlier in the NDD illnesses we studied, since we were restricted to CNS tissues from end-stage diseases.Our initial study of this approach yielded both very low frequencies of alignments .2.End-stage disease. We do not know the effects of end-stage disease, compared to earlier stages, on any of the variables we examined. For instance, we do not know about potential loss of infectious agents’ transcripts as disease progresses, nor do we know anything about expression of DAM genes over the course of illness in humans (compared to that in mice expressing mutated NDD genes).3.Dilution of microglial gene expressions. Microglia, if present, likely represent a minority of cells. As such, their contributions to total gene expression are predicted to be limited.4.Causal relationship(s) of Stage 1/Stage 2 DAM to neurodegeneration in each NDD. We do not presume to ascribe causality of Stage 1/Stage 2 DAM presence to the neurodegenerative process represented by the subjects who donated tissues we used. This is particularly of concern since we were not able to define clearly any potential causes for NDD phenotype or DAM gene expression.In spite of the above limitations, we hope that our findings will stimulate additional investigations into the potential role of DAM in pathogenesis of NDD’s. Lessening of DAM appearance or transition from Stage 1 DAM to Stage 2 DAM  may repr2 and PD (see text footnote 1) have been previously described. Briefly, tissues from persons with sporadic NDD’s were stored at -80 degrees and blocks dissected from these unfixed, frozen specimens. Frozen 20-micron tissue sections from these unfixed tissue blocks were placed into Qiazol buffer and stored at -80 degrees until RNA isolation was carried out . An on-column DNAase step was included for each sample. RNA quality was analyzed by gel electrophoresis. Illumina sequencing libraries were constructed according to manufacturer instructions and quantitated by qPCR, either by us  or by Cofactor Genomics  and paired-end (PE) Illumina sequencing was carried out by Cofactor Genomics, Inc.3 Compressed (gz) PE reads in fastq format were trimmed of Illumina sequence adapters (Trimmomatic®) and analyzed for expression based on the hg38 human genome by Tophat2/Bowtie2 (ALS) or HISAT2 . All graph constructions, correlations and statistical assays were performed in Prism 75.Our methods for tissue acquisition, RNA seq analyses and bioinformatics have been described in multiple publications . The parr HISAT2  and quanAll tissues were acquired commercially from National Disease Resource Interchange  ; under the auspices of an IRB-approved collection protocol (most AD/CTL and some PD/CTL), or were declared exempt from IRB oversight (some AD/CTL and some PD/CTL). All sequencing data discussed are the property of Neurodegeneration Therapeutics, Inc., and were acquired with private funds. Untrimmed, compressed (gz) FASTQ sequencing files are available to all legitimate investigators, following request to the corresponding author (JB), completion of a Material Transfer Agreement and provision of either a FTP URL or a memory storage device capable of storing 2 TB of data. Trimmed, processed BAM files following Trimmomatic and Tophat2/Bowtie2 analyses are also available upon reasonable request.JB, PK, and DB designed all the studies. PK oversaw the tissue acquisition and storage, and isolated and assayed the RNA’s. DB isolated the RNA’s (ALS samples), created and assayed the sequencing libraries (ALS), and performed the data analysis. JB performed all the bioinformatics assays and data analysis, and drafted the manuscript. All authors have reviewed the final manuscript and agreed with its contents.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Friend leukemia virus integration 1 (FLI1), an ETS transcription factor family member, acts as an oncogenic driver in hematological malignancies and promotes tumor growth in solid tumors. However, little is known about the mechanisms underlying the activation of this proto-oncogene in tumors.Immunohistochemical staining showed that FLI1 is aberrantly overexpressed in advanced stage and metastatic breast cancers. Using a CRISPR Cas9-guided immunoprecipitation assay, we identify a circular RNA in the FLI1 promoter chromatin complex, consisting of FLI1 exons 4-2-3, referred to as FECR1.Overexpression of FECR1 enhances invasiveness of MDA-MB231 breast cancer cells. Notably, FECR1 utilizes a positive feedback mechanism to activate FLI1 by inducing DNA hypomethylation in CpG islands of the promoter. FECR1 binds to the FLI1 promoter in cis and recruits TET1, a demethylase that is actively involved in DNA demethylation. FECR1 also binds to and downregulates in trans DNMT1, a methyltransferase that is essential for the maintenance of DNA methylation.These data suggest that FECR1 circular RNA acts as an upstream regulator to control breast cancer tumor growth by coordinating the regulation of DNA methylating and demethylating enzymes. Thus, FLI1 drives tumor metastasis not only through the canonical oncoprotein pathway, but also by using epigenetic mechanisms mediated by its exonic circular RNA.The online version of this article (10.1186/s13059-018-1594-y) contains supplementary material, which is available to authorized users. FLI1) was first identified as a proto-oncogene activated by proviral integration in F-MuLV-induced erythroleukemias [FLI1 is preferentially expressed in hematopoietic cells and tissues, where it regulates hematopoietic stem cell self-renewal and differentiation [FLI1 may act as a critical driver in the development of hematological malignancies [FLI1 in erythroblasts causes inhibition of differentiation and ultimately the development of pre-T cell lymphoblastic leukemia/lymphoma [FLI1 is also associated with other hematological malignancies, including pre-T cell lymphoblastic lymphoma, acute myeloid leukemia, and B cell lymphomas [The ETS gene family of transcription factors share a common ETS DNA binding domain responsible for sequence-specific DNA recognition on target promoters, and they play critical roles in controlling cell proliferation, transformation, apoptosis, and tumorigenesis –3. Amongeukemias . FLI1 isntiation , 6. Abergnancies –10. Overlymphoma . In addiFLI1 is also aberrantly expressed in some solid tumors, including Ewing sarcoma [FLI1 is overexpressed in breast cancers [FLI1 is strongly correlated with advanced stage, poor differentiation, and lymph node metastasis. In highly metastatic human breast cancer cells, knockdown of FLI1 significantly attenuated tumor metastasis through the Rho GTPase pathway [FLI1 expression score is associated with the stage of SCLC. In these cells, FLI1 promoted tumor metastasis by activating the miR-17-92 cluster family [Recent studies have shown that  sarcoma , 13, met sarcoma , nasopha sarcoma . A serie cancers  and lung cancers . In pati pathway . In non-FLI1 in these solid tumors. In this study, we harnessed a novel CRISPR Cas9-guided promoter immunoprecipitation (CasIP) assay to identify the molecular components that interact with the FLI1 promoter. We hypothesized that these components might actively participate in the control of FLI1 in the development of breast cancers. Using this CasIP assay, we identified FECR1, a FLI1 exonic circular RNA that binds to the FLI1 promoter and epigenetically activates FLI1 in breast cancer cells.However, little is known about the molecular mechanisms underlying the aberrant activation of FLI1 in breast cancer, we first examined its expression in tumor samples collected from patients with breast cancer. Using immunohistochemical staining, we found that FLI1 was significantly activated in breast tumor tissues as compared with adjacent normal tissues .We constructed a lentiviral vector containing the catalytically inactive CRISPR Cas9 (dCas9) and two FLI1 promoter was confirmed using CasIP PCR. We found that FECR1 was enriched in breast cancer cells that were transfected with Cas9-FLI1 gRNAs  assay , 19 to iter Fig. a. After FLI1 gene locus  to quantitate the expression of FLI1 in the FECR1-expressing cell clones. As seen in Fig. FLI1 as compared with the PBS and vector controls. Using Western blot, we found that overexpression of FECR1 significantly upregulated FLI1 in the treated cells  method to pull down TET1-interacting complexes. The pulled-down RNAs were reverse transcribed and quantitated by PCR using primers specific for FECR1. Using this assay, we detected an enrichment of FECR1 in the TET1 antibody-precipitated complex /2-oxoglutarate-dependent dioxygenase. TET1 is a critical factor that induces the oxidation-deamination mechanism underlying active DNA demethylation , 22. To lex Fig. c.FLI1 locus, we observed an enhanced binding of TET1 to the FLI1 promoter  in FECR1-overexpressing cells as compared with that in the vector control group. Taken together, these data suggest that FECR1 circular RNA may regulate its target genes, like FLI1, by binding to the gene promoter and recruiting TET1 DNA demethylase to induce DNA demethylation.To further confirm the interaction between FECR1 and TET1, we collected FECR1-overexpressing and vector control MDA-MB231 cells and performed chromatin immunoprecipitation with an antibody against TET1   sequencing, we demonstrate that this circular RNA binds to a variety of target genes that are involved in cell proliferation, transformation, apoptosis, and tumorigenesis. Notably, FECR1 binds to its own gene, FLI1, in cis and recruits TET1 demethylase. TET1 (ten-eleven translocation 1) is a member of a DNA hydroxylase family that possesses enzymatic activity toward converting 5mC into 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine through three consecutive oxidation reactions [FLI1. Thus, FECR1 activates a positive feedback mechanism which promotes oncogene transcription. Unlike the conventional FLI1 oncoprotein that regulates its downstream target genes, FECR1 functions by targeting the FLI1 gene transcriptionally via a cis mechanism.In this study, however, we have identified a new player in this malignant cascade. FECR1 circular RNA, a transcriptional byproduct of the proto-oncogene, promotes tumor metastasis at the level of gene transcription. FECR1 is a 571-bp circular RNA, consisting of eactions –32. By iDNMT1 promoter in trans. After promoter binding, FECR1 silences DNMT1 gene activity. In mammals, the genomic methylation process is executed by DNA methyltransferases DNMT1, DNMT2, DNMT3a, and DNMT3b [DNMT1. We predict that the downregulation of DNMT1 by FECR1 may cause activation of many target genes, including FLI1, that are associated with tumor growth. However, it is unclear how the binding of FECR1 induces downregulation of DNMT1. Future studies are needed to address the specific mechanisms by which FECR1 downregulates its downstream target genes, like DNMT1.We also demonstrate that FECR1 binds to the d DNMT3b , 33, 34.d DNMT3b , 35, 36.FLI1. If actively translated, it is predicted that FECR1 may produce a short truncated FLI1 (53 aa). Alternatively, it may use its internal ATGs as the initiation site and produces several short peptides. Future studies should validate the presence of these circular RNA-derived peptides and evaluate their functions in breast cancer.Circular RNAs belong to the category of long noncoding RNAs. However, recent studies have demonstrated that circular RNAs are also translated into functional peptides. With a combination of bioinformatic tools, Legnini et al. found that zinc finger protein circular RNA cir-ZNF609 is associated with heavy polysomes and is translated into a protein that controls myoblast proliferation . Using rEWS/FLI1 t translocation, although occurring in more than 80% of the cases with Ewing sarcoma/primitive neuroectodermal tumor (EWS/PNET), has not been reported in solid tumors. In the latter case, FLI1 is activated primarily by gene upregulation. In breast cancer, aberrant expression of FLI1 correlates with advanced stage, poor differentiation, lymph node metastasis, and disease-free survival [FLI1 attenuates tumor metastasis through multiple pathways, including the Rho GTPase pathway [FLI1 may function as a tumor enhancer or promoter in solid tumors. Future studies are needed to address whether the FLI1 gene may undergo subtle genetic alterations in solid tumors, including breast cancer.It should be noted that the survival , 39. Kno pathway  and the FLI1 contributes to tumor metastasis. We identified a novel FLI1 circular RNA FECR1 that interacts with the FLI1 promoter. FECR1 binds to the FLI1 promoter in cis and recruits TET1 demethylase. FECR1 also downregulates DNMT1 in trans. Together, this circular RNA may regulate metastasis of breast cancer cells by coordinating DNA methylation and demethylation in target genes that are involved in tumor growth. These data thus suggest that FLI1 circular RNA may serve as a potential therapeutic target in the development of therapeutic interventions for metastatic breast cancer.We have discovered a novel epigenetic pathway by which v/v) fetal bovine serum , 100 U/ml of penicillin sodium, and 100 μg/ml of streptomycin sulfate , at 37 °C in 5% CO2.Human breast cancer cell lines  and viral packaging 293 T cells were purchased from ATCC . Cells were routinely maintained in DMEM medium  containing 10% , 1 (weak), 2 (moderate), and 3 (intense). The immunoreactivity score was calculated as the percentage of positive cells per field multiplied by the intensity of staining.FLI1. The U6-gRNA1-T5-H1-gRNA2 cassette was synthesized by linking two Cas9-FLI1 gRNAs with the U6 promoter and H1 promoter. FLI1-gRNAs included FLI1-gRNA1 5′-GGGGTTGAGGACACGTGCTG-3′ and FLI1-gRNA2 5′-GAGCCAATATTCCGTAGCAT-3′. The expression cassette was ligated at the Pme I and Not I sites in the lenti CRISPR-EGFP sgRNA 2 vector (Addgene Plasmid #51761), in which the wild-type Cas9 was replaced with the catalytically inactive dCas9 , the supernatants were added in MDA-MB231 cells for transfection.Lentiviruses were packaged in 293 T cells using lipofectamine 3000  with 2 μg of lentiviral expression and packaging plasmids. Viral supernatants were collected at 24 and 48 h post transfection. After addition of polybrene  according to the manufacturer’s recommendations . BrieflyTotal RNA was extracted by TRIzol reagent  from cells and stored at − 80 °C. RT-PCR reaction was performed with an Eppendorf Thermol Cycler. The target amplification was performed by PCR of 1 cycle at 95 °C for 5 min; 33 cycles at 95 °C for 20 s, 60 °C for 15 s, and 72 °C for 20 s; and 1 cycle at 72 °C for 5 min. Quantitative real-time PCR was performed using 2× HotSybr Real-time PCR Kit . The threshold cycle (Ct) values of target genes were assessed by quantitative PCR in triplicate using a sequence detector  and were normalized over the Ct of the β-ACTIN control.FLI1 promoter gRNAs into the dCas9-2x gRNA vector that contains a mutated Cas9 (dCas9) and the tandem U6 and H1 promoters. Specifically, two oligonucleotides covering guiding RNA (gRNA) from the Fli1 promoter were synthesized and inserted immediately downstream of the U6 and H1 promoters, respectively, followed by the Cas9-hairpin RNA-(T)5 sequence.A Cas9-guided chromatin immunoprecipitation assay , 42 was The dCas9-Fli1 promoter lentivirus was produced in 293 T cells as previously described. The viral supernatants were filtered through a 0.45-μm filter, concentrated by a PEG-IT kit , aliquoted, and stored in a − 80 °C freezer. An aliquot of the Cas9-Fli1 promoter-gRNA lentivirus was used to transfect MDA 231 cells. The stable cells were selected in 1 μg/ml puromycin and collected for immunoprecipitation following the method as described previously. In brief, cells were fixed with 1% formaldehyde and sonicated for 180 s (10 s on and 10 s off) on ice with a Branson sonicator with a 2-mm microtip at 40% output control and 90% duty cycle settings. The sonicated chromatin TNAs containing dCas9-gRNA-Fli1 promoter complex were immunoprecipitated with Cas9 antibody . After reversal of cross-linking and proteinase K treatment, the Cas9-bound chromatin DNA and RNA were released and subjected to DNA/RNA sequencing and analyses.A RAT assay , 44 was To confirm FECR1 as a circular RNA, the RNA samples were treated with RNase R to remove the linear RNA , 45. Thebedtools genomecov function [Raw data were filtered with Fastx (version: 0.0.13), and approximately 12 million single-end reads of 50 bp length from RAT-seq were mapped to hg38 reference genome using bowtie2 (version 2.2.5) with default parameters . Only refunction . The resTotal cellular DNAs were extracted by Genomic DNA Purification Kit  and treated with sodium bisulfite using EZ DNA Methylation™ Kit  . BisulfiThe status of DNA methylation was determined by restriction enzyme digestion . GenomicFLI1 promoter and was performed using the method as described previously [ChIP assay was used to examine the binding of TET1 to the eviously . In brieeviously .RNA-CHIP was performed based on the protocol by Zhang et al. with minor modifications . MDA-MB2FLI1 and β-actin were obtained from Abcam .Cells were lysed in RIPA buffer in the presence of the protease inhibitor cocktail and 1 mM phenylmethylsulfonyl fluoride. Equal amounts of protein were resolved by SDS-PAGE and subjected to Western blot analysis using enhanced chemiluminescence (Pierce). Antibodies to t test. Comparison was made of groups with high FLI1 expression (score > median score) and low FLI1 expression (score ≤ median score). We assessed score comparisons between groups by one-way ANOVA test. Overall survival (OS) was calculated by using the Kaplan-Meier method, and the differences were assessed by using the log-rank test. p value of less than 0.05 was considered significant. Statistical calculations were performed using SPSS 13.0.Comparisons between groups were analyzed by Additional file 1:Figure S1.FLI1 overexpression in metastatic breast cancer tissues. Figure S2. Location of Cas9 gRNAs in the FLI1 promoter. Figure S3. Sequences of FECR1 variants. Figure S4. FECR1 from circular RNA database websites. Figure S5. Sequencing of Fli1 circle RNAs. Figure S6. FECR1 knockdown inhibited cell invasion in MDA-MB231 cells. Figure S7. Cellular distribution of FECR1 circRNA. Figure S8. Location of the FECR1-specific RAT primer. Figure S9. FECR1 binding by RAT-Seq. Figure S10. The chromatin RAT interactome of the FECR1 circRNA. Figure S11. FECR1 binding sequences in the CpG-rich FLI1 promoter. Figure S12. FECR1 enhances the binding of TET1 to the Fli1 promoter. Figure S13. Expression of target genes in FECR1-overexpressing cells. (PDF 2053 kb)Additional file 2:Table S1. Oligonucleotide sequences of PCR primers. (PDF 119 kb)