RNA origami is a recently developed RNA design approach that allows construction of large RNA nanostructures that can position aptamer motifs to spatially organize other molecules, including proteins. However, it is still not fully understood how positioning multiple aptamers on a scaffold and the orientation of a scaffold affects functional properties. Here, we investigate fusions of single-guide RNAs and RNA origami scaffolds (termed sgRNAO) capable of recruiting activating domains for control of gene expression in yeast. Using MS2 and PP7 as orthogonal protein-binding aptamers, we observe a gradual increase in transcriptional activation for up to four aptamers. We demonstrate that different aptamer positions on a scaffold and scaffold orientation affect transcriptional activation. Finally, sgRNAOs are used to regulate expression of enzymes of the violacein biosynthesis pathway to control metabolic flux. The integration of RNA origami nanostructures at promoter sites achieved here, can in the future be expanded by the addition of functional motifs such as riboswitches, ribozymes and sensor elements to allow for complex gene regulation.Designer RNA scaffolds constitute a promising tool for synthetic biology, as they can be genetically expressed to perform specific functions  A core goal of synthetic biology is the development of new molecular tools that enable complex gene regulation. Several approaches have been proposed, including the utilization of transcription activator-like effectors (TALEs), zinc-finger proteins and catalytically dead Cas9 (dCas9) regulation for both activation and repression of gene expression through the recruitment of activating or repressing protein domains . The advet al. extended the CRISPR-dCas9 system's regulatory capacity by designing sgRNA-based scaffolds (scRNA) which incorporate protein-binding RNA motifs capable of both locus targeting and additional regulatory action  several kilobases in length in sgRNAs   parameters generates RNA sequences that are predicted to have the desired folding characteristics ,24. RNA nX3′) ,28 were nX3′) . For cernX3′) .S. cerevisiae strain Y02569  provided by EUROSCARF. On all yeast strains, codon optimized S. pyogenes dCas9 is fused on the C-terminal with three tandem copies of SV40 nuclear localization signals (NLSs), while MCP-VP64 and PCP-VP64 are flanked with one N-terminal and one C-terminal copy of SV40 NLS. Open reading frames (ORFs) were taken from pJZC620  and sequences were adjusted for yeast MoClo toolkit compatibility , mVenus marker, and MCP/PCP expression cassettes  carrying VioABCDE under constitutive expression  cells (E. coli selection was performed in lysogeny broth (LB) agar plates containing either 34 μg/ml chloramphenicol, 100 μg/ml carbenicillin or 50 μg/ml kanamycin. Yeast transformations were performed using a LiAc/ssDNA/PEG protocol (B) cells . All bioprotocol . All yeaFlow cytometry analysis used an ACEA NovoCyte 2100YR system. Signal coming from mVenus was detected using a 488 nm laser for excitation and a 530/30 nm band pass filter. Data was captured using NovoExpress software measuring 10 000 gated events based on forward and side scatter to isolate singlets. Representative flow cytometry traces shown in −LEU− liquid cultures for 72 h at 30°C. For each strain, 2.5 ml of OD600 = 10 culture was then harvested and pelleted by centrifugation at 13 000 g. Supernatant was removed and cell pellet was resuspended in 500 μl methanol and lysed at 100°C for 20 min. Supernatant was then passed through a 0.22 μm filter before using it for HPLC analysis. Figure −LEU− plates for 72 h at 30°C. Cells were then resuspended in liquid SD-Glu URA−LEU− media and adjusted to OD600. 800 μl of OD600 = 8.3 culture was then pelleted by centrifugation at 13 000 g, and after supernatant removal pellet was resuspended in 200 μl methanol and lysed at 100°C for 20 min. Supernatant was passed through a 0.22 μm filter to remove cell debris. For HPLC measurements, either 200 μl   . Flow ra., 2013)  and D an., 2013) .S. cerevisiae yeast  and its Pol III promoter (tRNAPhep) sequence, separated by the self-cleaving hepatitis delta virus (HDV) ribozyme sequence. This strategy, which enables the use of native host promoters, has been shown to lead to improved sgRNA stability, while the HDV ribozyme ensures uncoupling from the translation machinery  transcriptional activation protein domain fused to the MS2 coat protein domain, were implemented , an mVenus reporter expression cassette under the very weak REV1 promoter (REV1p), sgRNAO expression cassettes following either the SNR52p or the tRNAPhep expression strategies and MCP-VP64 expression cassettes under constitutive TDH3 promoter (TDH3p) expression  was generated and one, two and three-MS2 aptamer sgRNAO derivatives  were created by replacing MS2 aptamers with tetraloop motifs. Figure To test whether our system is limited by the availability of MCP-VP64 ligands, in connection with an increasing number of MS2 aptamers on sgRNAOs which could explain 4xMS2-V_2’s performance, we attempted to express MCP-VP64 at either significantly lower or higher levels . IncreasFollowing the demonstration of sgRNAOs’ ability to carry multiple RNA protein-binding motifs and regulate expression, we opted to characterize the potential protein-binding aptamer positions of the 3-helix sgRNAO designs independently and identify which positions offer the best regulatory performance. Initially, we focused on the four positions used on the 4xMS2-V_2 sgRNAO  to the engineered loop  motif (4xMS2-3WJ) or horizontal to the engineered loop  were generated and compared with the 3xMS2-V  as well as enhance overall production. To achieve this, three new strains were created based on sgRNAOs carrying three copies of either the MS2 or the PP7 aptamers (3xMS2-V targeting RNR2p and 3xPP7-V targeting REV1p); GPY702 targeting PV production, GPY703 targeting PDV production and GPY713 targeting V production. Figure in vivo, demonstrating the incorporation of different functional elements. Here, utilizing the RNA origami scaffold technology previously described in in vitro experiments, we opt to create complex RNAs compatible with CRISPR activation thus proposing a powerful and modular molecular platform for synthetic biology applications. Fusions of sgRNA sequences targeting several genomic promoter regions with RNA origami-based scaffolds, recruiting several activating domains, are applied for transcriptional regulation. To achieve this, specific RNA design rules are applied, also considering host-related expression limitations. The significance of this method is further validated by the creation of complex RNA programs for the control of a branched metabolic pathway.The aim of this study is to verify that RNA origami can work as a scaffold Here, we have showcased that RNA origamis carrying protein-binding aptamers can be expressed in eukaryotes and successfully fold to enable transcriptional activation. Since sgRNAOs have to localize in the nucleus and scaffold RNA sequences can often be quite long, this imposed certain design limitations when attempting to express them from Pol III promoters. Therefore, sgRNAO sizes were kept to a minimum for the purposes of this study. Expression of sgRNAs from sequence-length resilient Pol II promoters has been shown in the past and several strategies to avoid downstream processing and ensure nuclear localization have been deployed, including the incorporation of self-cleaving ribozymes ,42. ThesTo exhibit compatibility of the system with different motifs, both the MS2 and the PP7 aptamers were selected and assessed within the CRISPR activation context. Scaffolding of multiple aptamers for multivalent recruitment of proteins was exhibited using sgRNAOs carrying up to four aptamers. The MS2 and PP7 aptamers have previously been shown to be orthogonal to each other even within the context of RNA scaffold-mediated CRISPR activation . From ouSpatial distancing dictated by RNA aptamer positioning and scaffold orientation appears to have an effect on promoter activation. This has also been previously demonstrated for scaffolded enzymes where small perturbations of a scaffold affect enzymatic activity . Our datThe design of sgRNAOs is dictated by multiple goals including its transcriptional efficiency within the cell, proper structure folding and optimal placement of the protein-binding motifs. Internal fusion of RNA scaffold sequence to the sgRNA, not only appears to increase stability of the overall structure but can also place scaffolded proteins closer to the promoter sequence potentially increasing regulation. Furthermore, internal fusion of RNA scaffolds essentially places the tracer RNA sequence of the sgRNA on the 3′ end of the overall sequence thus ensuring that only complete transcripts are capable of dCas9 binding. As a result, all transcripts that reach their genomic targets should be complete sgRNAOs and this also acts as a control of Pol III transcriptional capacity.in vivo for synthetic biology applications. RNA origami is highly modular and should allow the incorporation of other high-tier regulatory elements, such as RNA switches, enabling the creation of complex regulatory programs from single RNA scaffolds thus minimizing the number of RNA or other biological elements involved. With RNA being a simpler molecule to design and express in vivo than proteins, as well as imposing less burden, sgRNAOs can act as an important tool for synthetic biology and metabolic engineering.Overall, this work aims to set the foundation for the expression of large RNA origami scaffolds Data and materials are available upon request from the corresponding author.gkac470_Supplemental_FileClick here for additional data file.


Since the formalization of the Central Dogma of molecular biology, the relevance of RNA in modulating the flow of information from DNA to proteins has been clear. More recently, the discovery of a vast set of non-coding transcripts involved in crucial aspects of cellular biology has renewed the enthusiasm of the RNA community. Moreover, the remarkable impact of RNA therapies in facing the COVID19 pandemics has bolstered interest in the translational opportunities provided by this incredible molecule. For all these reasons, the Italian Society of Biophysics and Molecular Biology (SIBBM) decided to dedicate its 17th yearly meeting, held in June 2022 in Rome, to the many fascinating aspects of RNA biology. More than thirty national and international speakers covered the properties, modes of action and applications of RNA, from its role in the control of development and cell differentiation to its involvement in disease. Here, we summarize the scientific content of the conference, highlighting the take-home message of each presentation, and we stress the directions the community is currently exploring to push forward our comprehension of the RNA World 3.0. Summary: Review of the Italian Society of Biophysics and Molecular Biology (SIBBM) 17th yearly meeting, held in June 2022 in Rome, dedicated to the many fascinating aspects of RNA biology. The role played by RNA in regulating the information flow from DNA to proteins was formalized in 1956 with the formulation of the Central Dogma of Molecular Biology . HoweverGenetic information is transferred from DNA to RNA to protein, where RNA has been considered just an intermediary between DNA and proteins. The ‘RNA world’ became more and more complex in the latest 20 years with the discovery of non-coding RNAs, which have great importance in cellular processes. However, the border in the definition of coding and non-coding RNAs is quite blurry, since the two categories are interdependent. The two main functions of RNA, translation and structural role, could be associated to the different sub-cellular localization of the RNAs, since the translation takes place in the cytoplasm while the non-coding/regulatory functions are mainly exerted in the nucleus. Moreover, RNA molecules undergo alternative splicing, a process that could generate coding and non-coding isoforms from the same loci . New tecFor this reason, the Italian Society of Biophysics and Molecular Biology (SIBBM) decided to dedicate its 17th yearly meeting, held in June 2022 in Rome, to this fascinating aspect of RNA . The MeeIn this Meeting Review, we report on the current trends in RNA biology, focusing on their regulatory functions provided by (i) the structure of RNAs, (ii) their cellular localisation and (iii) the interactions with RNA binding proteins. An overview of the non-coding RNA and their implications with epigenomics regulation is also highlighted, focusing on circRNAs, lncRNAs and miRNAs. The SIBBM meeting drew attention to the newest technologies for RNA sequencing and analysis, such as direct sequencing, reserving a section of the meeting for describing state-of-the-art technology in this field.The conference welcomed about 270 participants, half of them junior researchers and graduate students. Here, we summarize the scientific content of the conference, highlighting the take-home message of each presentation, and we stress the directions which the community is exploring to push forward our comprehension of the RNA World 3.0.The first section of the conference, RNA structure, localization and RNA-protein interaction, was chaired by Valeria Poli and Francesco Nicassio. From the contributions of the speakers, a broad interconnection between non-coding RNAs (ncRNAs) and RNA binding proteins (RBPs) emerged. In this regard, the recently discovered concept of phase separation been particularly relevant across different biological contexts, with a particular impact on neuronal biology. RNA–protein interactions appeared to be modulated according to the structures (both primary and secondary) of the transcripts and emerged as a major factor driving RNA and protein localization.Fabrizio d'Adda di Fagagna , focused on the role played by ncRNAs in the DNA damage response, highlighting the contribution of his group to dissecting this mechanism. He presented a mechanism based on the synthesis and processing of long non-coding RNAs (lncRNA) at double-strand break sites. Through phase separation, the resulting transcripts mediate the recruitment and localization of proteins involved in DNA repair. Interestingly, since these RNAs are site-specific, their inhibition by antisense oligonucleotides (ASOs) allows the impairment of DNA damage signaling and repair at specific loci of the genome, providing an excellent resolution to investigate this process , who held the Armenise-Harvard Foundation Lecture, and whose research group is studying RNA localization. Specifically, the Baudet Lab used molecular beacon technology and advanced imaging approaches to characterize the transport of pre-miR-181a-1 in neurons’ compartments, suggesting that pre-miRNAs are shipped to distal axons by hitchhiking on late endosomes/lysosomes. In the growth cone central domain, where they are stored, pre-miRNAs are processed into newly generated mature miRNAs  in response to Semaphorin 3A. The mature miRNAs regulate the local translation of TUBB3, and impact axons’ growth and collapse. Overall, the results presented by Dr. Baudet not only reveal critical insight into the molecular mechanisms of ncRNA subcellular localization, but also provide fundamental knowledge of axonal transport; a process deeply involved in several neurodegenerative disorders  stayed on the subject, talking about the application of Basescope-FISH assays to study ncRNA localization in motoneurones. D'Ambra focused on the motoneurone-specific circular RNA (circRNA) circ-Hdgfrp3, which was found to traffic along neurites during cell maturation. This behaviour is impaired by oxidative stress, which causes a partial circ-Hdgfrp3 retention into stress granules. In motoneurones carrying FUS mutation, a condition linked to amyotrophic lateral sclerosis (ALS), the same stimulus results in the great segregation of circ-Hdgfrp3 into cytoplasmic mutant FUS assemblies. Interestingly, upon stress removal, the circular RNA is quickly released from stress granules but remains trapped into FUS-inclusions with the consequent impairment of its trafficking and function  who investigated the involvement of transposable elements Long Interspersed Nuclear Elements-1 (L1) in corticogenesis. L1-derived RNAs are required for regulating the balance between proliferation and differentiation in progenitor cells, and to establish the transcriptional program leading to neuronal differentiation and maturation. The data presented by Mangoni suggest an interaction of L1 RNAs with the Polycomb Repressive Complex 2 (PRC-2) to regulate the deposition of the repressive epigenetic mark H3K27me3 on PRC-2-target genes. This study position L1 RNAs as crucial signaling hubs for genome-wide chromatin remodeling, enabling the fine-tuning of gene expression during brain development.Neuronal development was also the focus of the contribute by Gian Gaetano Tartaglia , was invited to present the work of his group on RNA-mediated phase separation. First, he presented novel algorithms able to integrate local properties of protein and RNA structures to quantitatively predict long RNA binding proteins partners. These technologies enabled the Tartaglia Lab to reveal the association of ncRNAs (e.g. Xist) with proteins involved in the regulation of transcriptional and translational programs, as well as neurodevelopmental (FXTAS) and neurodegenerative diseases (Parkinson's disease)  . Furtherdisease) .Mihaela Zavolan , was invited to give the Human Technopole Lecture. She presented the work of her group on 3′ untranslated regions (3'UTRs) and alternative polyadenylation (APA). She gave an overview of the methodologies and resources developed by her lab to profile polyadenylation signals and 3'UTR isoforms, as well as RBP regulators of 3′ end processing. Moreover, she presented very interesting applications of her methodologies to the investigation of APA in cancer. For instance, she introduced approaches to infer poly(A) sites usage (PAQR) and motifs responsible for this regulation (KAPAC), showing their application to identify a key regulator of APA in glioblastoma (PTBP1). She also presented a method (SCUREL) suitable to quantify changes in 3′ UTR length between groups of cells. This tool was applied to investigate the differential alternative polyadenylation between malignant and infiltrated cells in lung adenocarcinoma, and it revealed a general trend toward 3′ UTR shortening for cells in the tumor microenvironment  versus normal counterparts. In general, these techniques proved to be extremely informative to investigate 3'UTR isoforms, with potential repercussions on several human pathologies including cancer and haematological, immunological and neurological diseases  presented her study on the arginine methylation of RNA binding proteins. She profiled RBPs–RNA interaction through Orthogonal Organic Phase Separation after the pharmacological inhibition of protein arginine methyltransferases (PRMTs), and during cisplatin-induced replicative stress. Indeed, even if previous studies from her group have demonstrated that cisplatin treatment could change PRMT subcellular localization, her results suggest a novel layer of complexity. In response to the cisplatin treatment the R-hypomethylation on RBPs, the consequent modulation of RBP-RNA binding and their subcellular localization can occur  presented an example of riboregulation describing his work on the structural characterization of the complex between cytosolic serine hydroxymethyltransferase and its cognate RNA modulator. These are the first 3D structural data of a riboregulated enzyme. His results suggest that the RNA molecule acts as a conformational switch, allosterically regulating the enzyme and locking it in a conformation unfavorable for the binding of the substrate serine. This observation provides insights on how RNA can allosterically control the activity of metabolic enzymes, and it offers an approach for future RNA-mediated therapies based on the targeting of these enzymes  presented the work on motoneuronal phenotypes upon ALS FUS mutation. He showed that the cytoplasmic localization of mutant FUS perturbs the RBP network which regulates the activities of important RBPs (HuD/ ELAVL4 and FMRP). Specifically, mutant FUS competes with FMRP for the binding of HuD 3′UTR and results in its upregulation. Consequently, NRN1 and GAP43, which are targeted by HuD, are stabilized and induce increased axon branching and growth upon injury; a phenotype which can be rescued by dampening NRN1 levels  are the most abundant type of non-coding transcripts that are more than 200 nucleotides in length. They are classified in different subtypes, such as intergenic lncRNA, intronic lncRNA, bidirectional lncRNA, antisense lncRNA and enhancer RNA (eRNA). RNA polymerase II is generally responsible for transcription of lncRNAs. Similar to mRNAs, lncRNAs undergo splicing, 5′ ends capping and 3′ ends polyadenylation processes . LncRNAsIgor Ulitsky  shed light on some questions regarding the functionality of lncRNA, their maturation process and evolution. Understanding whether their functions are modulated and correlated to disease progression could improve the therapeutic use of lncRNA. In particular, Ulitsky focused on the role of Silc1 lncRNA expressed during neurodegeneration. His group demonstrated that Silc1 lncRNA activates in cis the expression of Sox11, hence its depletion in vivo leads to a delayed regeneration in a tissue specific manner  focuses research on the mechanisms underpinning the recruitment of RNA binding proteins (RBPs) by lncRNAs and their trafficking from the nucleus to the cytoplasm. Mendell highlighted the role of a lncRNA activated by DNA damage (named NORAD), a highly conserved and cytoplasmic RNA responsible for genome stability in mammals. NORAD binds Pumilio RBP and the loss of NORAD leads to Pumilio over-activation. Hence, Pumilio downstream targets (mitosis and DNA repair regulators) are repressed, causing genomic instability in mice. A genome-wide screening strategy was employed to investigate the mechanism underlying NORAD nuclear export. This new study shed light on the role of RBM33, a RBP able to mediate the nuclear transport of several transcripts included NORAD, demonstrating that RBM33 binds GC-rich elements in target transcripts. The NORAD-Pumilio pathway and the RBM33 preference for the GC-rich regions can be translated to other transcripts to shed light on the regulation of the interactions between RBPs and lncRNAs and the mechanisms for GC-rich transcript transport , aimed to unveil which lncRNAs bind the transcriptional coactivator protein p300. As results of a PAR-CLIP interaction screening, they identified about 120 lncRNAs actively interacting with p300 in embryonic stem cells. Among these, lncSmad7 binds the C-terminal domain of p300 and is responsible for maintaining the stem cell self-renewal. In conclusion, lncSmad7 recruits p300 in order to regulate genome regions in trans, in particular genes involved in the development  discussed the X Chromosome inactivation (XCI) from the RNA interactome point of view. As known, XCI is essential for female development, hence it becomes of great interest to investigate the mechanisms underpinning the kinetics of X-linked gene silencing, discriminating among the regulatory elements of certain genes and dissecting the spatial organization of the genome. She gave new insights into the regulation of X inactive-specific transcript (XIST), the lncRNA which acts as trigger and master regulator of XCI. The question about the link between dosage compensation and epigenetics remains open as well as the understanding of XCI escape that leads to disfunction of female development and sexual dimorphism  is a major expert on lncRNAs and their regulation. His group developed the cap analysis gene expression (CAGE) tool that has the dual goal to identify the transcription start sites (TSSs) of capped RNAs and measure transcripts expression levels , focuses her research on the regulation of gene expression involved in the maintenance of T-cell quiescence. The study draws the attention on the LINE1 family of transposable elements that are found in the group of genes significantly regulated during the function and exhaustion of effector T-cells. In particular, the regulation of LINE1 transcripts relies on the transcriptional factor IRF4, while Nucleolin is responsible for their maintenance in the chromosome. LINE1 family transcripts affect the deposition of H3K36me3, acting in cis on the genome region where they are. When T-cells are activated and differentiated, LINE1-transcripts are downregulated by means of splicing suppression mechanisms. Interestingly, strong accumulation of LINE1-transcripts has been found in dysfunctional T-cells, while the depletion of LINE-1 transcripts is able to restore the function of the T-cells and revert the exhaustion. These results could be exploited from a clinical perspective, where the T-cell exhaustion could be addressed modulating LINE1 transcripts with antisense oligonucleotides (Non-coding transposable elements (TEs) or transposons represent a class of interesting transcriptional regulators, previously defined as ‘jumping genes’, due to their ability to move from one region of the genome to another. Their sequences are strongly conserved. LINEs and SINEs are one of the largest components of the family, mostly located in introns. The LINE1 family has more than 500,000 copies in the human genome. leotides .Manuel Beltran Nebot,  shed light on a new the interaction between circRNAs and mRNAs that can be a targetable mechanism to treat diseases. In particular, CircZNF609 is upregulated in many cancer types and its modulation reduces the tumor aggressiveness, acting as an mRNA sponge. Among the specific pairs of CircZNF609 and mRNAs, it is interesting to observe the one with CKAP5 mRNA, which encodes for a protein that regulates microtubules, involved in processes such as cell migration or the organization of the mitotic apparatus. When the interaction between circZNF609/CKAP5 mRNA was inhibited by means of small interfering RNAs (siRNAs) against circZNF60, the cells showed a higher sensitivity to drugs targeting the microtubules, such as vincristine or paclitaxel. This strategy shows an interesting potential for the control of cancer cells growth and in long term it will improve cancer treatments (Circular RNAs (circRNAs) are covalently closed circular RNA molecules, deriving from a biogenesis process where a downstream 5′ splice site of an exon is joined to an upstream 3′ splice site of the same or another exon. CircRNAs can interact with proteins and other RNAs, acting as microRNA sponges, can regulate transcription and they can also be translated in proteins. Recently, circRNAs gained attention because they have been found in biofluids and are under study as possible biomarkers for the detection of pathological states . Little eatments .Marta Biagioli  focuses her work on Huntington's disease, a genetic autosomal dominant neurodegenerative disorder, still lacking a pharmacological treatment. It has been shown that trinucleotide CAG repeat expansion in the HTT gene is the most frequent mutation in Huntington's disease. The triplet expansion leads to a longer sequence of glutamine tract at the amino terminus of the huntington protein, impairing its structural properties and functional activities. Biagioli described that the CAG expansion in HTT correlates with alterations in splicing events in neural progenitors. The work sheds light on new possible mechanisms that are dysregulated in Huntington's disease and the regulatory interactions with miRNAs and RBPs  presented his group's research in Acute Myeloid Leukemia. They performed a CRISPR-Cas9 dropout screen in mouse leukemia cells and among the targets for cell proliferation they found an enzyme involved in 7-methylguanosine cap hypermethylation (TGS1). Though RNA-IP-Seq experiments in different human AML cell lines they identified more than 500 mRNA targets carrying trimethylguanosine on their cap structure that are highly enriched in cellular respiration and oxidative phosphorylation pathways. Importantly, TGS1 knockdown in acute myeloid leukemia results in the downregulation of oxidative phosphorylation (OXPHOS) with a consequent oxidative stress. Overall, Barbieri's group identified TGS1 enzyme as a master regulator of cellular oxygen metabolism in AML that could act as potential therapeutic target in leukemia.We are witnessing the emergence of the epitranscriptomics era. This field studies reversible chemical modifications of bases within an RNA molecule and is now taking medical research by storm since this post-transcriptional marks impact cell fate and development. There are more than 100 RNA modifications and recent studies demonstrate that many enzymes responsible for regulating RNA modifications could be ideal targets for cancer therapy . Isaia BAnders H. Lund  presented the work of his lab and showed that rRNA modifications are a source of ribosome heterogeneity, impacting normal development and pathology  is interested in how chromosomal rearrangements drive tumorigenesis. His group has used CRISPR-Cas9 technology to induce specific chromosomal rearrangements in vivo and ex vivo  aims at investigating the possible implication of N6-methyladenosine (m6A) modification in the molecular mechanism of SINEUP activity  gave an overview of the impact of m6A modulation in chronic myeloid leukemia (CML). He showed that the m6A methyltransferase complex METTL3/METTL14 is upregulated in CML patients, and its knockdown inhibits cell proliferation. Furthermore, his group elucidated the consequences of METTL3 altered subcellular localization, showing that cytoplasmic METTL3 has a role independent from its nuclear catalytic activity. In fact, the knockdown of METTL3 in the cytoplasm affects translation in CML and he hypothesized that the translation-promoting effect on methylated mRNAs could represent a general mechanism for cells where METTL3 is localized in the cytoplasm  is the most prevalent, conserved, and abundant internal RNA modification of mRNAs in eukaryotes. m6A functions as a critical regulator for gene expression and is dynamically and reversely controlled as an analogy to the epigenetic code formed by DNA and histone modifications . Accumulytoplasm . MoreoveAna Boskovic  investigates the molecular mechanisms that allow epigenetic information to be transmitted through gametes from parents to offspring in mammals. Previous work from her postdoc lab demonstrated that protein restriction of male mice modulates levels of various small RNAs in mature sperm. They showed that tRNA fragments are the most abundant small RNAs in sperm and, in particular, the 5′-end fragment of tRNA Glycine-GCC (5′tRF-GG) is consistently upregulated in response to low-protein diet in mice  focuses her research on the adaptive responses of triple-negative breast cancer (TNCB) cells. She developed a novel approach to search and identify functional lncRNA loci. This new method relies on the selection of candidate lncRNAs obtained by high-resolution transcriptional and epigenomic studies, followed by their functional characterization with CRISPR interference (CRISPRi). She was able to build a set of 620 lncRNA-TNBC candidates that were targeted in a CRISPRi library which was employed in parallel dropout screenings for different adaptive phenotypes. With this approach she identified about 100 lncRNAs involved in cancer phenotypes, including 3D growth, in vivo tumorigenesis or adaptation following drug treatment. Interestingly only few lncRNA loci were found as involved in unchallenged 2D proliferation, suggesting a specific role for lncRNAs in proliferation or survival upon challenging conditions. This represents a reliable approach aimed at identifying lncRNA loci that are relevant in cancer and provide new insights into the regulatory roles that lncRNAs can exert in cancer cell plasticity  presented her work focused on mitotic kinase Aurora A (AURKA), a member of a family of mitotic serine/threonine kinases that is essential for cell proliferation. In the last few years different studies reported its kinase-independent activity in cancer cells  who spoke about mRNA translation in cancer. Many tumors share an immune escape strategy called tryptophan degradation, where a shortage of tryptophan is generated upon interferon-γ (IFNγ) induction. In this pathway, tryptophan is transformed into kynurenine by the enzyme indoleamine 2,3-dioxygenase 1 (IDO1), which then inhibits the function of T cells, leading to immunosuppression. The Agami lab has investigated further the effects of prolonged IFNγ treatment on mRNA translation in melanoma cells by ribosome profiling. Agami's data show a global reduction in mRNA translation upon treatment and that, because of tryptophan shortage, ribosomes skip tryptophan codons by going out-of-frame and producing aberrant proteins  is also focused on cancer, but from the microRNA (miRNA) perspective. Specifically, his lab works on diffuse large B-cell lymphoma (DLBCL), which accounts ∼30%–58% of all non-Hodgkin lymphomas (NHL). In this landscape, recently miR-155 emerged as a diagnostic marker as well as a therapeutic target . Since miR-155 was found to be overexpressed in patients with ABC-DLBCL in comparison with healthy controls, the Slack's group delivered cobomarsen to DLBCL cell lines, monitoring the binding of the drug with the endogenous miR-155, with a miR-155 biosensor. Cell proliferation and apoptosis were followed as outcome measurement of the cobomarsen treatment: in vitro results showed decreased proliferation and increased apoptosis of DLBCL cells. In addition, when the drug was administered in vivo, they observed reduced tumor growth and no adverse events, demonstrating that the drug was well tolerated . One patient experienced positive results. Despite the first positive results, the patient was discontinued from the cobomarsen clinical trial after 21 total doses of cobomarsen through five cycles, because of disease progression. Cobomarsen/anti-miR-155 targeting shows some promise in the clinic, but further refinements are necessary. Another project in the Slack's lab concerns miR-34. The miR-34 family is transcriptionally induced by p53  focuses his research on static and dynamic RNA structure elements. The Incarnato lab’s aim is to develop strategies that overcome the technical challenges of identifying highly relevant RNA structure elements in living cells. The identification of putative functional RNA structure is of crucial importance, especially when it comes to RNA therapeutics. There are many softwares that predict the RNA structure based on free energy minimization, when the RNA sequence is given, but the biologically active structure is not necessarily the most stable. Because of this limitation, Incarnato's lab developed DRACO, an algorithm for the deconvolution of coexisting RNA conformations from mutational profiling experiments. They recently applied DRACO to SARS-CoV-2 genome, which identified some regions that fold into two mutually exclusive and different conformations  talked about the impact of ALS-related FUS mutations on the subcellular RNA localization and membrane-less compartments composition. It is well known that FUS mutations are responsible for the incorrect localization and cytoplasmic aggregation of the protein, which causes toxic condensates, affecting all cellular functions  presented her research on triple-negative breast cancer (TNBC), a tumor responsible for up to 20% of all breast cancers, known to be difficult to treat, because the high chance of relapse , speaker invited to hold the Riccardo Cortese Lecture, who shared the work of her group on the study of RNA as therapeutic molecule. Her lab works on Spinocerebellar ataxia type 3 (SCA3), known also as Machado Joseph disease, caused by CAG triplet expansion in exon 10 of the ATXN3 gene. This expansion causes a toxic gain of function, leading to neurodegeneration. They designed four different antisense oligonucleotides (ASOs) that were then transfected in SCA3 patient-derived fibroblasts, to induce exon 10 skipping. Three out of four ASOs did induce exon 10 skipping producing a shorter ataxin-3 transcript and truncated protein. Moreover, they tested these ASOs in vivo, in MJD84.2 mouse model, a transgenic mouse model containing a human ATXN3 gene with expanded CAG region. The efficacy of the ASOs was similar to what observed in SCA3 patient-derived fibroblasts. Surprisingly, the ASOs had a long-lasting effect in mice, without affecting bodyweight or motor behavior , a nonprofit consortium, whose mission is the development of RNA therapeutics for patients with very rare diseases. The first RNA-drug customized for one single patient was already designed and produced in 2019. Milasen was, indeed, a splice-modulating ASO drug, designed at Boston's Children Hospital for Mila, a child affected by a rare and fatal neurodegenerative condition , the father of Spinraza, without any doubt the most successful and groundbreaking ASO-based approved therapy so far. One of the present antisense projects in the Krainer lab concerns pediatric high-grade gliomas (pHGGs), one of the most malignant tumors. Among different mutations annotated, one very common in this type of tumor was found in the gene encoding for histone H3.3, where methionine replaces the lysine in position 27 (K27M), altering the epigenetic status of the cell. This event could then lead to tumorigenesis , that showed how U1 small nuclear RNA (snRNAs) can be modified as desired, to obtain chimeric antisense RNA to induce target-specific exon skipping with therapeutical purposes. She worked on mutations in retinitis pigmentosa GTPase-regulator (RPGR) gene that cause X-linked retinitis pigmentosa, a subtype of retinal dystrophy. An intronic mutation in intron 9, affects the splicing of RPGR gene, increasing the production of RPGR-transcripts containing the pseudoexon 9a (E9a) by a factor of ∼3.5. When E9a is present in the mature mRNA, a premature stop codon is introduced causing the production of a truncated protein. To restore the impaired gene expression, she designed some modified U1 antisense snRNAs molecules which target specifically E9a in RPGR mRNA. In vitro experiments demonstrated that three chimeric U1 snRNAs efficiently mediate E9a skipping, restoring RPGR transcript expression patterns , presented his work that describes the consequences of global inhibition of the microRNA pathway during tumor development. The approach used in this study relies on the inducible expression of a small protein, named T6B, able to interfere with the assembly of the microRNA-induced silencing complex (miRISC). In the first part of the talk, he discussed evidence that different tissues contain a diverse population of AGO complexes, mainly composed by low molecular weight RISC (LMW-RISC) and high molecular weight RISC (HMW-RISC) complexes. The latter conceivably represent the pool of miRISC engaged in target repression, and can be induced by the occurrence of mitogenic cues  gave a talk about inference of transcriptional and post-transcriptional dynamics from sequencing experiments. Gene expression is determined by RNA synthesis, processing and degradation, each with a kinetic rate. Because transcriptional and post-transcriptional modulations leave specific footprints on premature and mature RNA temporal profile, this can be exploited to infer kinetic rates. Furlan developed INSPEcT, anovel approach based on the mathematical modeling of premature and mature RNA expression. This computational method can quantify kinetic rates from steady-state or time-course total RNA-seq data, without requiring any information on nascent transcripts  platforms have revolutionized molecular biology due to their exceptional throughput and accuracy, however, they are sub-optimal for specific purposes. For example, they provide short reads which typically cover only a small fraction of the original molecule, and this complicates tasks like genome assembly and transcriptome profiling. Moreover, the retro-transcription steps intrinsic in sequencing protocols introduce biases and preclude the direct sequencing of original RNA fragments preventing direct nucleotide modifications detection. Novel sequencing platforms have been developed in the last few years to overcome these limitations, one of the most relevant is the sequencing provided by Oxford Nanopore Technology (ONT). This technology allows direct DNA and RNA sequencing of full-length molecules and represents an optimal solution to the limitations of NGS platforms. Since its release, Nanopore sequencing has deeply impacted (epi)genomics and (epi)transcriptomics, and this session was dedicated to present examples of how this technology was applied across different biological contexts.Camilla Ugolini  used Nanopore direct RNA sequencing to investigate SARS-CoV-2 virus complexity. Direct sequencing (DRS) provides remarkable advantages for isoform detection, but DRS suffers of some limitation for the resolution of the sequences at the 5′ cap, as sequencing proceeds in the 3′ to 5′ direction and starts from the end of the transcript . To overcome this problem, Ugolini used a new approach, termed as Nanopore ReCappable Sequencing (NRCeq). In NRCeq the native 5′ cap is replaced with a 5′ cap-linked RNA sequencing adapter, allowing to discriminate full-length, capped molecules from fragmented RNAs and truncated sequencing artefacts. With this new approach Ugolini was able to profile the repertoire of full-length, capped RNAs produced by the SARS-CoV-2 virus and, hence, to identify novel canonical and non-canonical sgRNAs. Overall, this work provides important insights into the mechanisms that regulate the transcription of SARS-CoV-2 sgRNAs  was selected to present ribonucleotides (rNMPs) detection through Nanopore DNA sequencing. The strategy used to characterize rNMPs on Nanopore sequencing is based on detecting sequencing errors and current intensity signals, revealing characteristic patterns for strands containing rNMPs. This strategy was applied on the circular ssDNA of M13 phage containing ribonucleotides at specific positions. Primers complementary to the M13 phage were designed and extended using the ssDNA as template, and the resulting double strand DNA molecules were linearized and sequenced. By means of state-of-the-art bioinformatic tools, Dr. Fonzino was able to reveal the characteristic patterns for strands containing rNMPs. This preliminary work supports the feasibility of rNMPs detection from nanopore sequencing data and suggests the possibility for interesting advancements with potential applications in the study of genome stability.Sìlvia Carbonell Sala  from Guigó Lab tackled the problem of the characterization of the noncoding transcriptome. Since the old dogma of coding RNAs has been questioned, more and more noncoding transcripts have been discovered. However, the great majority of these RNAs (>97%) remain functionally uncharacterized. This problem is partially due to the lack of a complete annotation, with many gene models being fragmented and/or uncatalogued. As part of the GENCODE consortium, the Guigó lab recently developed a new method, capture long-read sequencing (CLS), to improve accuracy and completeness of the lncRNA catalogue . This prize was awarded in loving memory of Professor Riccardo Cortese, a proactive member of the international scientific community who provided outstanding basic and translational results in the field of gene expression.The 2022 “Francesca Martini” prize for the best poster on cancer research went to Francesca Orso , who presented a contribute focused on the role of the miRNA miR-214 in mediating the crosstalk between stroma and malignant cells during tumor progression. This prize was awarded by NAnA onlus, a non-profit organization created in loving memory of Francesca Martini to actively support the promotion of scientific research on prevention and treatment of tumors.The 2022 “Finally, the following participants were selected for the best poster awards: Chiara Barzan , Davide Colaianni , Stefano Cretti, , Dario Dattilo , Alessandro Ferrando , Silvia Piscitelli , Francesca Priante  and Daniele Viavattene .After two years of online conferences, the 17th annual meeting of the SIBBM has been a fresh new start for the scientific community. Overall, it represented a great opportunity to discuss about new frontiers in the RNA world. Among the many topics touched during the conference, the relevance of RNA beyond protein synthesis emerged as particularly relevant in developmental process and in many diseases including cancer and neuro-diseases. New concepts like RNA modifications, RNA structure, RNA localization and phase separation are also proving to be crucial in the field and possibly interconnected. Novel techniques, mainly for imaging and sequencing, were also introduced along with the exciting insights they might provide in all the aforementioned topics, and into coding and non-coding RNA functions across physiological and disease conditions. For a remarkable number of young speakers, the SIBBM conference has been their first opportunity to present their work in front of an outstanding audience of 270 scientists. Together with the two poster sessions, this resulted in an intense scientific discussion, palpable during the three days of conference, which will impact the future research activities on the RNA field, both in Italy and across borders.


Podarcis siculus has represented a suitable organism for the study of spermatogenesis over the years. In this lizard, the process of spermatogenesis is regulated by the interaction between systemic factors such as gonadotropins and local factors, i.e., molecules produced by the somatic and germinal cells of the testis. Many exogenous substances are able to alter the production of these regulative factors, thus altering the course of spermatogenesis, and P. siculus has proven to be an excellent model for studying the effects of various endogenous or exogenous substances on mechanisms underlying spermatogenesis. This review summarizes the available data on the effects of different substances on the control of spermatogenesis, highlighting the induced morphological and molecular alterations. Overall, the data show that sex hormone levels as well as the final stages of spermatogenesis are most affected by an imbalance of endogenous compounds or contamination by environmental pollutants. This is helpful for the male individual, since the damage, not affecting the spermatogonial stem cells, can be considered transient and not irreversible.Spermatogenesis is a genetically driven differentiation process that occurs in the testis and leads to the formation of spermatozoa. This process is extensively studied in several experimental models, particularly in vertebrates that share the morphological structure and functionality of the mammalian testis. Although reptiles are not generally considered biological models, the lizard  Podarcis siculus   are the most abundant  axis, which in turn is controlled by the photothermal regime. In P. siculus, mating and ovulation are confined to a brief period every year. Between late April and mid-July, females lay 12 to 20 eggs in 2–3 episodes about 20 days apart [In eterothermal species living in temperate areas, such as ys apart . Clutch P. siculus, in fact, gametes development takes a year to be completed. In females, follicles are recruited in fall, remain quiescent in winter, and start vitellogenesis in early spring [The initiation and modulation of reproductive processes, however, are timed earlier, by predictive proximate factors represented by seasonal changes in day length. In y spring . In the y spring , leadingP. siculus is believed to possess an endogenous reproductive rhythm, by virtue of the observations that populations belonging to the same species, despite experiencing different conditions of daylight and ambient temperature at different latitudes, breed in the same period of the year [P. siculus males kept in constant darkness at 5 °C or in daily photophases of 14 h at 28–30 °C from late summer onwards showed a testicular recrudescence in the following March at about the same period of the naturally occurring event [Seasonal changes in day length, therefore, act as a zeitgeber entraining the endogenous circannual rhythm ,15. P. sthe year . It has ng event .In this lizard, testes have a typical tubular organization that changes during the annual cycle determining six different conditions ,18,19,20P. siculus represents one of the main animal models used to study the intricate process of spermatogenesis since the 1950s, as evidenced by the studies of Galgano and D’Amore [P. siculus, many of which helped to elucidate the spermatogenic process in other vertebrates that share a similar testicular organization, including mammals. It has been shown that the process is regulated by a network of interaction between several factors of both systemic and testicular origin [P. siculus testis of these molecules with their corresponding receptors both in the germline and in somatic cells, suggesting their involvement in the control of testicular activities [PACAP and VIP mRNA levels during the different phases of the reproductive cycle, with the lowest mRNA expression levels recorded during the summer and winter stasis, when spermatogenesis is blocked; the highest transcript levels were detected during the reproductive period, characterized by maximum testicular activity; finally, intermediate expression levels were found in the recovery phases, when spermatogenesis is reactivated [PACAP and VIP mRNA levels are directly linked to changes in P. siculus testis activity. Furthermore, in situ hybridization and immunocytochemical investigations have shown that PACAP, VIP, and their receptors are widely distributed in the Podarcis testis throughout the reproductive cycle and localized in both germ cells and somatic cells [P. siculus. D’Amore . Over tir origin ; among tr origin ,26,27,28r origin ,30, betar origin ,32, retir origin , and ster origin ,35 desertivities ,25,26. Ictivated ,25,26. Tic cells . Taken tP. siculus of beta-endorphin (β-EP), an endogenous opioid peptide involved in the control of reproductive processes in vertebrates, has also been demonstrated [In addition to neuropeptides, the presence in the testis of nstrated ,32,36,37nstrated .RAR α and RAR β) during the different phases of the reproductive cycle. In the reproductive period (May), only RAR β is expressed, while in the post-reproductive phase (August) only the transcript for RAR α is present [In another study, it was shown that an additional molecule such as vitamin A and its main biologically active derivative, retinoic acid (RA), is involved in the control of lizard spermatogenesis . Indeed, present .P. siculus, the expression of the Vasa gene, which encodes a protein involved in different aspects of germ cell development in many animals [More recently, through biochemical and molecular investigations in the testis of  animals , has bee animals ,41. UsinP. siculus spermatogenesis is also played by steroidogenic enzymes, which represent the key to managing the local action of sex hormones on spermatogenesis, whose localization and levels have been studied  [P. siculus testis throughout the reproductive cycle have been established by immunocytochemistry; this analysis showed that, in lizard testis, as occurs in mammals [A major role in the control of  5α-Red) ,35. The  mammals ,43,44, s mammals ,35.The balance between cytochrome P450 aromatase expression and sex hormone levels is really interesting: the highest levels of P450 aromatase, the enzyme responsible for estrogen biosynthesis, and 17β-estradiol were recorded during the summer and winter stasis when the hormone is involved in blocking the spermatogenesis ,28,45; cP. siculus have played an important role in the field of reproductive toxicology, as they have led to the identification of many toxic substances capable of altering spermatogenesis, also clarifying the molecular mechanisms underlying the toxic effect in many cases.Dysregulation of the factors controlling spermatogenesis severely impairs sperm production and, consequently, reproductive success. The studies carried out on It has been demonstrated that the balance and cooperation between the factors controlling spermatogenesis can be severely compromised by overexposure to the same physiological factors, as well as by many environmental molecules, such as herbicides and pesticides, xenoestrogens, and endocrine disruptors (EDCs).P. siculus can be modified by the administration of molecules of endogenous nature, with a consequent alteration of their levels in the testis; obviously, any substance capable of modifying the concentration of these factors in the organism has a spermatogenesis effect.The spermatogenesis of P. siculus, a high constitutive level of D-Asp was found in the testis, showing cyclical variations in quantity during the annual reproductive cycle in parallel with sex hormone levels. Administration of exogenous D-Asp showed that the lizard testis was specifically able to take up and accumulate this amino acid, leading to increased testosterone levels in both the testis and plasma. Exogenous D-Asp also resulted in a significant increase in mitotic activity of the testis by inducing spermatogenesis, recognized by an intense immunoreactivity of the germinal epithelium (spermatogonia and spermatids) for the proliferation cell nuclear antigen (PCNA) [P. siculus testis has been well documented [In vitro studies carried out on testis organ cultures have demonstrated that the addition of both PACAP and VIP to the culture medium causes the overproduction of sex hormones such as testosterone and 17 β-estradiol; consequently, it has been hypothesized that these local factors may cooperate with each other and act on the transcription of enzymes involved in the production of hormones such as P450 aromatase. This hypothesis is supported by the fact that the gene encoding for this enzyme has a response element to the second messengers released upon the interaction of neuropeptides with their receptors ,28,34,35n (PCNA) . Differecumented , has beecumented .P. siculus suggested a role for estrogens in modulating spermatogenesis in this organism as well, and the observation that the inhibition of P450 aromatase induced by fadrozole treatment impairs lizard spermatogenesis confirms this hypothesis [P. siculus males regulate spermatogenesis by blocking it during testicular quiescence and eliminating supernumerary germ cells through apoptosis during the reproductive phase [Another important player in spermatogenesis is the steroid hormone estrogen, resulting from the aromatization of testosterone. Although estrogen is generally recognized as a female sex hormone, numerous studies demonstrate that it plays a fundamental role in controlling spermatogenesis . The abipothesis . In partve phase ,48.However, as with other endogenous substances, overexposure to estrogen, or to estrogen-like substances, can induce harmful effects, with consequent alteration of the regulation of the spermatogenic process. For example, it has been demonstrated that 2 days of exposure to exogenous estradiol caused an increase in spermatogonial cell proliferation through activation of the ERK1/2 signaling pathway . The proVTG gene can be activated by exposure to estrogen or estrogen-like substances [VTG, the major precursor of yolk proteins, is an estrogen-dependent and sex-specific protein synthesized in the liver of the females of oviparous vertebrates during the reproductive period . In malebstances . For thiP. siculus were obtained using anti-estrogenic or estrogenic substances, such as tamoxifen or clomiphene. The first can reduce the Il-mediated increase in mast cell number in the testis and in the Harderian gland [Other data that emphasize the importance of a correct balance of estrogen during the male reproductive cycle of an gland . Clomiphan gland . The expan gland . Clomiphan gland . All theP. siculus lizards, a wildlife animal living in fields and, therefore, potentially exposed to them. In a study performed in 2008 [The rapid growth in the use of pesticides and herbicides for agricultural production around the world has prompted many researchers to study the effects of these substances on  in 2008 , it was  in 2008 .P. siculus, causing sterility. Indeed, the morphological analysis demonstrated that acute treatment with this substance induced a massive reduction of germ cells in all stages of differentiation, the appearance of multinucleated giant cells, and the reduction of the lumen of the seminiferous tubules. Chronic treatment was even more harmful, inducing a significant decrease in germ cells with the appearance of vacuoles in the seminiferous epithelium and the disappearance of the lumen. In addition, the molecular analysis demonstrated a dose- and time-dependent decrease in the expression of androgen and estrogen receptors following treatment with MT [Cardone in 2012 showed that another substance, the fungicide methyl-thiophanate (MT), altered the spermatogenesis of  with MT .Another chemical compound that has similar effects on lizard spermatogenesis is the insecticide imidacloprid. Acute and chronic exposures have shown that this substance causes a reduction of the spermatogenesis process together with alterations in plasma levels of sex hormones and in the intratesticular expression of mRNAs for androgen and estrogen receptors .P. siculus spermatogenesis at both molecular and histological levels [ERβ.More recently, it has been demonstrated that the herbicide glyphosate has negative effects on l levels . In detaImmunocytochemical analysis showed that the alteration of the estrogen/estrogen receptor system causes an alteration in the structure of the seminiferous tubules by changing the distribution of connexin 43, a testis-specific gap junction protein that connects Sertoli cells and germ cells, the expression of which is regulated by estrogen. The absence of connexin 43 results in a disruption of the seminiferous epithelium, characterized by the presence of holes between the germ cells that aggregate forming structures termed rosettes. The authors also demonstrated that glyphosate induces the deposition of connective tissue between the seminiferous tubules, leading to fibrosis  Figure , a proceEndocrine disrupting compounds are chemicals that mimic, block, or interfere with hormone activity. They are associated with various dysregulations of the body’s endocrine system, causing adverse effects in cells and tissues ,62,63. GP. siculus, the testicular secretion of testosterone could be inhibited by the alkylating sulfonic ester ethane 1,2-dimethane sulfonate (EDS). Research carried out during two different moments of the reproductive cycle (winter stasis and breeding period) showed that exposure to EDS caused a reduction in androgen levels due to the impairment of Leydig cells, the main source of testosterone in the testis [A first study dated 1995 demonstrated that, in e testis .P. siculus via food and water and was shown to cause a slowdown in spermatogenesis through a striking structural alteration of the testis and epididymis, making them similar to those of the non-reproductive period. NP treatment has also been shown to inhibit the expression of AR, ERα, and ERβ genes in spermatogonia and primary spermatocytes and to shut down the secretory activity of the epididymal corpus by inducing ERα expression [The effect of nonylphenol (NP), an endocrine disruptor with estrogen-like properties, was also investigated. In a first series of experiments, NP was administered to pression .P. siculus have been evaluated [More recently, the effects of nonylphenol alone and in mixtures with another endocrine disruptor, octylphenol, on the reproductive capacity of valuated . The datvaluated  (Figure In conclusion, the data collected here show that the exogenous administration of endogenous substances that control spermatogenesis, such as neuropeptides, amino acids, and hormones, as well as any exogenous substance capable of dysregulating their production, leads to structural and functional changes in the testis. A more deleterious effect on the morphology and functionality of the seminiferous tubules was recorded following the administration of substances that directly mimic endogenous hormones such as EDCs, insecticides, and herbicides.P. siculus spermatogenesis.In particular, the changes concern the increase/decrease in the levels of estrogen, testosterone, androgen and estrogen receptors, the inhibition of the aromatase enzyme, the reduction in the levels of pituitary gonadotropins, as well as the thinning of the epithelium of the seminiferous tubules, and the loss of connections between germ cells and Sertoli cells. Interestingly, the morphological analysis also showed that the greatest damage found in the seminiferous tubules of differently treated males was in the reduced number of spermatids and spermatozoa, thus demonstrating that the latter are the most sensitive stages of germ cell differentiation. While this result indicates that the exogenous substances, in addition to altered levels of endogenous ones, lead to infertility due to lack of spermatozoa, on the other hand, it demonstrates that the interruption of these perturbative effects could restore the efficiency of the seminiferous tubules and the production of spermatozoa, since they do not irreversibly affect spermatogonial stem cells.Hence, the results allow us to predict the loss of reproductive capacity of these animals, as their testis is very sensitive to the action of environmental contaminants. Sharing testicular morphology and spermatogenic activity with many other terrestrial vertebrates, including mammals, these results are also predictive of any damage to the reproductive health of all species exposed to these substances. This once again demonstrates the lizard’s ability to be an excellent but unconventional biological model.


A body of work spanning neuroscience, economics, and psychology indicates that decision-making is context-dependent, which means that the value of an option depends not only on the option in question, but also on the other options in the choice set—or the ‘context’. While context effects have been observed primarily in small-scale laboratory studies with tightly constrained, artificially constructed choice sets, it remains to be determined whether these context effects take hold in real-world choice problems, where choice sets are large and decisions driven by rich histories of direct experience. Here, we investigate whether valuations are context-dependent in real-world choice by analyzing a massive restaurant rating dataset as well as two independent replication datasets which provide complementary operationalizations of restaurant choice. We find that users make fewer ratings-maximizing choices in choice sets with higher-rated options—a hallmark of context-dependent choice—and that post-choice restaurant ratings also varied systematically with the ratings of unchosen restaurants. Furthermore, in a follow-up laboratory experiment using hypothetical choice sets matched to the real-world data, we find further support for the idea that subjective valuations of restaurants are scaled in accordance with the choice context, providing corroborating evidence for a general mechanistic-level account of these effects. Taken together, our results provide a potent demonstration of context-dependent choice in real-world choice settings, manifesting both in decisions and subjective valuation of options. A classic example of choice behavior shaped jointly by presumed processing limitations and the demands of the decision environment is the context-dependent nature of preferences: the relative value of an option depends not only on the option in question but the other options in the choice set, or context4. This sort of context-dependent valuation explains a number of interesting patterns of choice both in humans9 and animals10.A central theme in the study of decision-making is the limited-capacity nature of human information processing11 which posit that value is a static quantity independent of the context, and accordingly, preference between two options should be independent of contextual factors such as the other available options. At the same time, relative (versus absolute) valuation could be understood, computationally, as a rescaling of reward values, relative to the other values present in the context, allowing an individual’s representation of value to adjust to the current value context5. Indeed, recent theoretical treatments have argued that contextual—versus absolute—value representations permit a decision-maker to discriminate between two low-value options in one context, and at the same time, two high-valued options in another context13.Context-dependent—rather than stable—valuation famously challenges classical theories of rational choice15—and in tightly constrained, small-sample demonstrations of “independence of irrelevant alternatives” (IIA) violations17. While a body of work has examined context effects arising from choice set composition—probing how the distribution of option values faced by the decision-maker systematically alter choices and subjective valuations of options—limited work has investigated whether these sorts of context effects hold in real-world settings18. Unlike tightly-controlled laboratory settings, the choice sets facing real-world decision-makers are arbitrarily large, and composed of options, which could be valued along multiple dimensions, each learned via rich histories of direct experience. Beyond the possible practical implications of any real-world context effects—which hold sizable economic consequences both for consumers and retail businesses—understanding how these real-world context effects unfold is important for understanding the boundary conditions of these well-studied laboratory effects. For example, it is possible that these sorts of context effects do not scale to more naturalistic settings, where individuals may resort to heuristics (e.g. ‘taking the best’). Supporting this view, more naturalistic presentation of options has been shown to reduce apparent suboptimalities in choice19.Context effects have been observed in both real-world choices—with respect to the temporal history of information viewed, or past choices faced by decision-makers23. Accordingly, we reasoned that we could leverage large-scale datasets that reflect real-world choices in diverse settings in order to glean novel insights about how context effects play out ‘in the wild’25, thus directly evaluating whether these effects are meaningful and general26. Here we elucidate whether context effects could be observed in actual choices with real-world consequences—for both the decision-maker and the restaurant28—at a scale and breadth sufficient to permit detection of context effects while averaging over potential option- and subject-specific effects.More generally, our understanding of context effects in choice might be inherently limited by the artificial, but well-constrained nature of tasks employed in these laboratory choice studies29 and (2) the user’s subjective valuation of their consummatory experience30, this rating data permits large-scale examination of contextual effects on both inferred choices and valuations. In our analysis, we view each restaurant decision as one made from a “choice set”—defined as similar, geographically proximate restaurants—with the goal of choosing the restaurant with the highest utility32.We demonstrate the generality of context-dependent valuation—both in choices and self-reported valuation—by analyzing a massive real-world restaurant rating dataset. Here we examine user behavior on Yelp, a web-based application which maintains crowd-sourced reviews of local businesses which is particularly popular in North America. Our analysis specifically focused on restaurants because, in most cases, people visit one restaurant per meal—ensuring that options effectively compete with each other—and the categories of restaurants are relatively well-defined in our dataset. With the presumption that each Yelp user’s rating evidences both (1) a decision made to visit the rated restaurant over some competing set of options22, we hypothesized that we would see fewer choices to the highest-rated option in higher-valued contexts . One mechanism underpinning such a choice context effect would be if options were selected on the basis of expected values, which are themselves context dependent. To shed light on these intermediate computations we performed a second, laboratory-based study, testing the hypothesis that if decision-makers’ subjective valuations are context dependent, then their ratings of the expected quality of a given restaurant would also exhibit context dependence in accordance with the value distribution of the choice set, even in the absence of explicit choice. Taken together, we find evidence for context-dependent decision-making and valuation in real-world behavior  and furthermore, laboratory-based evidence suggesting that these context effects in choice and subjective valuation are closely related.Our large-scale real-world analysis of ratings addresses two empirical questions. First, what is the impact of the local value context upon choice efficiency?—namely, do Yelp users make less ratings-maximizing choices in sets with higher overall ratings? Based on laboratory results indicating that choice contexts with more valuable ‘distractor’ items  decrease individuals’ propensity to choose the highest-valued itemhttps://www.yelp.com/dataset): (1) reviews of businesses, for which each record consists of a unique user ID, a unique business ID, the date and time of review, the unique ID of the business reviewed, the user’s given star rating (ranging from 1 to 5 in whole numbers), and the text of the user’s review, and (2) data describing each business, which consists of unique business IDs, business names, GPS coordinates (latitude and longitude) and restaurant categories. We note that the reviews in this dataset reflects the output of Yelp’s recommendation software, which filters out suspect or biased reviews on the basis of quality, reliability, and user activity patterns.Real-world data analysis focused on two data sources in the Yelp Open Dataset  with at least 1000 restaurants, leaving six US MSAs in the analysis: Charlotte-Concord-Gastonia NC-SC, Cleveland-Elyria OH, Las Vegas-Henderson-Paradise NV, Madison WI, Phoenix-Mesa-Scottsdale AZ, and Pittsburgh PA (US), and two Canadian MSAs: Calgary AB, and Toronto ON. We only examined choices pertaining to restaurant businesses—that is, all business entries containing the category “Restaurants” in the Yelp data, which totaled 59,315 restaurants in the US and Canada. Each business was mapped to an MSA with the US Census Bureau’s defined Core-Based Statistical Areas (in the US) or Census Metropolitan Areas (in Canada). We examined user reviews that were posted between January 1 2012 and November 14 2018 .As business in the Yelp dataset are assigned multiple, unordered categories , we inferred the single category that best described each restaurant. For each restaurant, we tabulated the occurrence of each Yelp-assigned category term in the text of the user reviews associated with that restaurant, and used the most frequently-occurring category term (e.g. “Pizza”). This ensured that each restaurant occurred in at most one possible consideration set, defined by restaurant category and neighborhood, preventing a restaurant from occurring in multiple consideration sets .34, a density-based spatial clustering algorithm that permitted us to define “clusters” based on users’ restaurant visit patterns, allowing neighborhoods of any arbitrary shape. The DBSCAN algorithm, implemented in the scikit-learn package (https://scikit-learn.org) for Python35, took the GPS coordinates of each restaurant review as input  and two parameters: a search radius , and a minimum number of data points within the search radius. Because the restaurant density and overall size of the MSAs considered vary considerably, the search radius and the minimum number of points were separately tuned for each MSA by performing nearest-neighbor analysis on the restaurants’ GPS coordinates and the total number of restaurants in each MSA, following previous work36. Across all MSAs, the average number of restaurants, irrespective of category, per neighborhood  was 11.389  MSA for each user, for which we assume users likely have the most knowledge of the restaurant options. The resultant distribution of users per MSA was as follows: Phoenix-Mesa-Scottsdale AZ: 29.84%, Las Vegas-Henderson-Paradise NV: 28.33%, Toronto ON: 21.26%, Charlotte-Concord-Gastonia NC-SC: 7.382%, Pittsburgh PA: 4.849%, Cleveland-Elyria OH: 4.324%, Calgary AB: 2.078%, and Madison WI: 1.935%.27. Our analyses also took into consideration the number of ratings present for the highest-rated option in the set, and the price (expressed the number of dollar signs from ranging from 1 to 4) of the highest-rated option in the set. We computed the probability of choosing the (possibly non-unique) highest-rated restaurant in the choice set, following previous work22—as a function of the mean star rating of the choice set in question computed over these rounded star ratings.For every restaurant that a user reviewed, we analyzed that restaurant in the context of the restaurant’s category and its inferred geographic cluster—which, together, define a choice set. Our analysis only considered choices made within sets composed of 3 or more restaurants, with at least 3 unique ratings , resulting in 53,118 choices examined in the final analysis. For each option in the choice consideration set, we computed the mean star rating for each option on the date of the user’s review—that is, considering reviews up to the point of that review but not including the review to capture the user’s possible informational state at the time of choice—rounding to the nearest half-star, as is seen by Yelp usersIn our analyses of post-choice ratings, the Rating Deviation measure was computed as the difference between the mean rating of the chosen restaurant before that user’s rating and the user’s rating itself. To ensure user ratings measured pure context effects, the average star rating of the choice sets  if their current GPS coordinates are proximate to the coordinates of the business they are checking into. Unlike ratings, the aggregate number of check-ins to a business are not visible to other users, which mitigates the possibility that a check-in could be used to bias the rating distribution or popularity of a particular restaurant.M = 8503; note these are not linked to specific users), which we analyzed similarly to the ratings-inferred choice analysis. For the 565,661 check-ins considered in the analysis, which spanned the years 2014–2016, we determined the surrounding choice set for that check-in using the same neighborhood and category definitions as in the ratings-based choice analysis and tabulated whether that check-in was made to the highest-rated option in that choice set. We then examined whether the mean star rating of the choice set influenced the probability of checking into the highest-rated option.The Yelp dataset contains a list of timestamped check-ins for each business , the number of previous ratings for the restaurant at the time of the order, and the price of the restaurant. Here, we defined choice sets as unique combinations of cities and cuisine types . We filtered out choice sets with less than 5 restaurants, leaving 821 unique choice sets in total. We calculated the probability of a user ordering from the highest-rated restaurant, for each choice set separately over the course of the two months. Mirroring our analysis of the Yelp datasets, we then examined whether the mean star rating of each choice set influenced the user’s probability of ordering from the highest-rated restaurant in the set.In our second conceptual replication dataset, we analyzed choice patterns of customers ordering food on Deliveroo, an online food delivery service popular in several countries outside of North Americahttps://cran.r-project.org/web/packages/MCMCglmm/index.html) for R37. In our regression models, all fixed effects were also taken as random effects over users (or participants). The resultant estimates of these fixed effects are reported in the regression tables. In all applicable models, we log-transformed the length of the text review, the number of reviews for the chosen option, and the number of options in the current choice set to mitigate skew apparent in these variables.In both the real-world and laboratory data, we estimated mixed-effects regression models (using logistic regression in models predicting choices) using Markov chain Monte Carlo with uninformative priors using the MCMCglmm package .We examined signatures of context-dependent choice and subjective valuations using a dataset from Yelp, a website and smartphone app popular in North America, on which users review local businesses such as restaurants and retail stores. Each rating is made on a scale of one to five stars, with higher star ratings indicating a better rating. For every Yelp user considered, we analyzed each user-provided restaurant rating—which, following previous work39. Critically, this analysis considered each option’s average rating at the time of the user’s presumed choice—rounded to the nearest half-star, as is presented to users on Yelp—in order to capture the decision-maker’s information state at the time of restaurant selection.This choice set was defined as all restaurants belonging to the chosen restaurant’s category within the same neighborhood (e.g. “Pizza” in a particular neighborhood in Pittsburgh). These geographic neighborhoods were defined by a density-based spatial clustering algorithm, taking all user reviews as input Fig. ” sectionβ = 0.5182, 95% CI 0.4145–0.6149, p < 0.0001; Table We first sought to establish that users’ choices were sensitive to option ratings, as well as the ratings of alternatives in the choice set. Consistent with the idea that our operationalization of choice and definition of choice sets capture a value-comparison process, we observed that the likelihood that a user chooses the highest-rated option (e.g. a 4.5-star option) increased as a function of the difference between the highest-rated option and the second-highest-rated option  in the choice set , again reflecting the well-documented set size effect . Importantly, our analysis controlled for the number of reviews40 and the price of the highest-rated option31 by taking them as covariates in the regression. Similarly, this context effect remained significant  even when controlling for the price of the second highest-rated option, for which we found that more expensive second-highest-rated options predicted less likely choices to the highest-rated option . Furthermore, this context effect—that, the negative effect of mean choice set star rating upon ratings-maximizing choice—was statistically significant even when controlling for the variance of choice set star ratings  or using different geospatial definitions of choice sets .We next examined whether the value context—i.e. the star rating composition of the choice set—affected individuals’ propensity to choose the highest-rated restaurant. We found that as the mean star rating of the choice set increased, choices of the highest-rated option  decreased markedly , we sought to rule out the possibility that the apparent suboptimality in making ratings-maximizing choices in sets with higher average option ratings .Importantly, this analysis takes a user’s rating as a proxy for their choice to visit a restaurant, but we sought to demonstrate that these choice context effects take hold in two independent replication datasets which operationalize choice differently. First, we examined a larger dataset of ‘Check-ins’ on Yelp, which are time-stamped, geolocated records of a user having visited a particular restaurant, using the same choice set definition as the previous analysis . Examining users’ propensity to ‘Check into’ the highest-rated option as a function of the mean star rating of the choice set Fig. B, we fou31). As in the two other Yelp datasets, we again found a marked context effect . Taken together, these two independent replication datasets support the generality of these context effects of across different settings and operationalizations of choice.As another means of conceptual replication, we examined whether context effects could also be observed in actual purchase decisions from the online food delivery service Deliveroo , a dataset recently used to understand exploratory behavior in consumer choice , examining the ratio of choices made between the two top-rated options as a function of the quality of the “distractor” items inferior to the two highest-rated options. This analysis provides a more direct examination of possible violations of independence of irrelevant alternatives (IIA), according to which the choice ratio between two options should not depend on the quality of other “irrelevant” options41. Importantly, this analysis only considered the subset of choices made to the highest- or second-highest-rated options in each choice set , probing whether the relative ratio of choices made to the best option varied as a function of the average ratings of the distractor items . Conceptually replicating Louie et al.22 and echoing the results of our prior analysis, we observed that choice sets with more valuable inferior options led to significant decreases in the relative ratio of ratings-maximizing choices in both ratings-based choices , indicating that higher-rated restaurants were more likely to receive higher star ratings after being chosen. Taking this dependency into consideration, we focused on predicting the deviation between the user’s rating and the average restaurant rating at the presumed time of choice . Importantly, our regression model controlled for a number of variables including the number of previous reviews, the price of the chosen option, and average previous rating of the chosen restaurant  contexts.Interestingly, the Yelp ratings examined here—which reflect a user’s consumption experience with a particular restaurant—also afford the opportunity to probe how choice context modulates a user’s subjective valuation of an option following a presumed choice. Intuitively, a user’s restaurant ratings correlated with the average star ratings left by other users before the current rating was made . More formally, we conjecture that the subjective valuation of an option, reflected in a user’s rating of that option, may be computed as the difference between the experienced utility of the restaurant and the contextually-computed expectation of that option (akin to a “prediction error”45). On this view, choice contexts with higher average ratings drive should reduce the user’s expectation of an option, in proportion to the average rating of the choice set. Assuming that the user’s experienced utility for an option is a symmetric random variable centered around that option’s ground-truth utility , an option rated in a higher-valued context should receive higher ratings relative to the options’ ground-truth value. Indeed, this exact pattern manifests in the rating deviations . This sample size, which we have adopted as a standard for online studies employing within-subject designs, ensures adequate statistical power to detect meaningful differences across conditions, while at the same time protecting against false positives results or inflated effect sizes which can result from small samples. Experimental protocols were approved by the McGill University Research Ethics Board (REB) and the methods described hereafter were carried out in accordance with REB guidelines. Participants provided informed consent in accordance with the McGill University REB and were paid $4.00 USD for their participation. Thirty-eight participants were excluded from the sample due to failing to correctly respond to at least 60% of catch trials (see below), leaving a final sample size of 62 . Note that the exclusion of these participants does not affect the significance of results reported below.We collected data from 100 participants on Amazon Mechanical Turk, using the psiTurk packageOur experiment utilized 275 choice sets extracted from the real-world analysis described above. Each choice set was presented to participants in a 4 × 3 grid of restaurants depicting an image of each restaurant (from the yelp website), the average star rating of each restaurant , the number of reviews used to compute the average star rating, the price of each restaurant represented as dollar signs ranging from one to four, and the category . Finally, each participant completed 5 catch trials (used to identify participants who did not follow instructions), in which they were required to choose the restaurant with the highest star rating. Importantly, no specific directions were provided concerning the information participants should use to make choices and ratings.For all trial types, the available options were presented on the screen for 5 s during which time participants could not make a response, and following this viewing period, participants had 5 s to make a response. Following each choice and rating, participants rated their confidence in the previous response using a that ranged from 1  to 7 (“Very confident”). Participants had 5 s to make a confidence response. Confidence data were not examined in the present analyses.Following the real-world dataset, inferential statistics for choices and expected satisfaction ratings were analyzed with mixed-effects regressions, taking all predictor variables as fixed and random effects at the participant level. Participant-level effect sizes in choice and rating context effects were computed as per-subject regression coefficients from the group analysis (conditioned on the group level estimates), superimposed on the estimated group-level effect.β =  − 0.8538, 95% CI − 1.321, − 0.4892, p < 0.0001; Table β = 0.0116, 95% CI − 0.2272, 0.2906, p = 0.462). In other words, our laboratory experiment replicated the choice context effected observed in the real-world choice behavior—more valuable contexts were associated with less likely choices of the highest-rated option.Analyzing choices, we observed participants were significantly less likely to choose the highest-rated ‘target’ option in choice sets with higher mean ratings . In other words, the degree of context-dependence in an individual’s choices strongly related to the degree of context-dependence in their ratings, supporting the generality of these context effects to both choice and subjective valuation.Finally, we probed the relationship between individual differences in the extent of context-dependence manifested in choice—quantified by the subject-level effect of mean choice set rating upon the probability of ratings-maximizing choice—and context-dependence evident in separately-measured expectation ratings. To do this, we examined context-dependence in expectation ratings separately for participants with small versus large context effects in choice  of the options alone, suggesting that this real-world rating information was evaluated—and acted upon, insofar as directing users’ choices—in a relative and context-dependent, as opposed to absolute, manner.Here we examined whether real-world choices and valuation, evidenced in a large dataset of online restaurant reviews, are subject to context effects that have previously been observed in tightly-constrained laboratory settings22. Taken together, the marked context dependence we observe ‘in the wild’ and experimentally further buttress the idea that an option’s value is computed relative to its context, an idea pervasive across neuroscience, psychology, and behavioral economics47. Computationally, relative—versus absolute—value representation holds the benefit that information processing resources are allocated efficiently to the encoding of subjective values13.Further corroborating this account, in a laboratory experiment, we found evidence for context dependence in decision-makers’ choices and subjective expectations of options’ values, which, beyond replicating real-world choice context effects in a controlled setting, demonstrates that the value composition of a choice set can systematically alter decision-makers’ expectancy of an option’s value. Further, the concordance between the extent of context-dependence observed in individuals’ choices and context-dependence in ratings Fig. C further48). Future work should examine, more directly, the extent to which subjective (consumption) utility might also exhibit context dependence.Our results raise a number of questions about the nature of these observed context effects. While our account postulates that the expectations informing the real-world ratings are computed in a relative, context-dependent fashion—which finds more direct support in our laboratory experiment—an open question concerns whether the subjective utility of consumption is also computed contextually, relative to the value composition of the choice set (or some other reference point). However, such effects could also arise from processes operating at consumption  are informed by ratings users observe on the Yelp website (or smartphone app) at the time of presumed choice, it may be the case that these online ratings may act here as a proxy for users’ knowledge about the value of options in the environment, rather than directly informing the observed choices. In other words, some users’ (absolute) valuations of options could be informed by direct experience with options or ‘word of mouth’ rather than star ratings. While user ratings are often a valid—if noisy—indicator of “true” quality of the options, we remain agnostic about which source(s) of information are the basis for these choices. The qualitative concordance between real-world choice patterns Fig. A and lab31, or that, in choosing across different categories, a restaurant could present as an option in multiple choice sets . However, any such construction would presumably be orthogonal to the context effects observed here, absent any asymmetric relationship between restaurant rating and category membership. More directly, it is unlikely that these simplifying assumptions systematically hold the consequence that these robust context effects observed are the result of artifacts in light of the laboratory-based replication of these choice patterns. Nevertheless, future work could endeavor to understand how choice set rating distributions of different categories (and possibly even other choice sets of the same category) might bear on context-dependent choices and ratings. Finally, while a follow-up analysis revealed that these context effects were robust to the spatial definition of choice sets , we should note it is possible that individuals might, at times, strive to choose the more globally “optimal” restaurant with respect to an entire city (or large region of a city) rather a single neighborhood per se.It also is worth noting that our analysis approach—as in any real-world choice study —relies critically on the operationalization of choice sets, which in our case were spatially-defined clusters of restaurants belonging to a single category. This simplified our analysis by guaranteeing that each restaurant appears in at most one choice set, and that restaurant options only competed with each other within the context of a single category. Of course, it is possible that an individual considers multiple restaurant categories when choosing between restaurantsSupplementary Information 1.


D., Espeland, M. A. & Langefeld, C. D. Nature Communications 10.1038/s41467-021-21038-1 (2021)1, highlighted the importance of accounting for the dependence between cells from the same individual when conducting differential expression analysis on single-cell RNA-sequencing data. Their work proved the inadequacy of pseudoreplication approaches for such analysis—this was an important step forward that was conclusively proven by them. However, there appear to be limitations in both their benchmarking and simulation approaches. Here, we corrected these issues, reran the author’s analysis and found that pseudobulk methods outperformed mixed models. Based on these findings, we recommend the use of pseudobulk approaches for differential expression in single-cell RNA-sequencing analyses.Recently, Zimmerman et al.1, performed a systematic analysis of differential expression methods’ type-1 error rates; the proportion of non-differentially expressed genes indicated as differentially expressed by a model. Their analysis was conducted on simulated, single-cell expression data across 20,000 iterations. The authors tested iterations of 5–40 individuals and 50–500 cells using an unadjusted p-value cut-off of 0.05 for significance. Plotting the results showed pseudobulk approaches had the lowest type-1 error at every iteration  curves for different proportions of differentially expressed genes.We tested the models using the Matthews Correlation Coefficient (MCC) giving a balanced measure of performance. MCC is a well-known and frequently adopted metric in the machine learning field, which offers a more informative and reliable score on binary classification problems3 and agrees with Zimmerman et al.’s findings1. On the other hand, both pseudobulk approaches; ‘Pseudobulk: Mean’ and ‘Pseudobulk: Sum’, showed improved performance as the number of cells increases. This trend was also noted in two of the other models; ‘GEE1’ and ‘Tweedie: GLMM’.Our MCC analysis demonstrates that pseudobulk approaches achieve highest performance across individuals and cells variations Fig. . There iMoreover, for statistical test comparisons, another approach is to compare the power of tests at the same test size. That is, to compare the models’ sensitivity 1–type-2 error) at a consistent type-1 error rate. Therefore, we generated ROC curves for the different approaches, enabling such comparisons. For example, in Supplementary Fig.  error atp-value cut-off of 0.05, they benchmark the performance of different methods at identifying non-differentially expressed genes (Supplementary Fig. p-value cut-off. Given this conservative 95% confidence intervals of pseudobulk methods, they could, as a result, have a higher type-2 error than other methods. However, our ROC analysis disproves this. It shows how, at equal type-1 error rates, pseudobulk mean has the lowest type-2 error rate of all tested methods (Fig. Zimmerman et al. argued that pseudobulk methods are “overly conservative” relative to mixed models in their work. Specifically, they refer to pseudobulk approaches’ lower than nominal levels of type-1 error rates, demonstrated in their results where, based on a consistent 4. To mirror this, we simulated data with an imbalanced number of cells between case and controls. We find that pseudobulk mean outperforms all other approaches on this analysis (Supplementary Fig. 5. This approach was taken by Zimmerman et al. as their data are simulated one independent gene at a time without considering differences in library size. The effect of this step is more apparent on the imbalanced number of cells where pseudobulk sum’s performance degrades dramatically. Pseudobulk mean appears invariant to this missing normalisation step because of the averaging’s own normalisation effect. Importantly, this was a flaw in the simulation software strategy and does not show an improved performance of pseudobulk mean over sum. We believe this approach also affected the performance of pseudobulk sum on the different proportions of differentially expressed genes (Fig. All analysis to this point have been on simulations with an equal number of cells in each sample. However, in real datasets this would never be the casenes Fig. .6. Notably, the pseudobulk method used here, DESeq25, performed worse than other pseudobulk models in Squair et al.,’s analysis and so their adoption may further increase the performance of pseudobulk approaches on our dataset. Conversely, Squair et al., did not consider all models included in our analysis or the different forms of pseudobulk aggregation. Therefore, our results on sum and mean pseudobulk extend their findings and indicate that mean aggregation may be the best performing. However, the reader should be cognisant that the lack of a normalisation step based on the flaw in the simulation software strategy likely causes the increased performance of mean over sum aggregation. Further, the use of simulated datasets in our analysis may not accurately reflect the differences between individuals that are present in biological datasets. Thus, despite both our results and those reported by Squair et al., there is still room for further analysis, benchmarking more models, including different combinations of pseudobulk aggregation methods and models, on more representative simulated datasets and biological datasets to identify the optimal approach. Specifically, we would expect pseudobulk sum with a normalisation step to outperform pseudobulk mean since it can account for the intra-individual variance which is otherwise lost with pseudobulk mean, but this should be tested, including on imbalanced datasets and at consistent type-1 error rates.Pseudobulk approaches were also found to be top performing in a recent review by Squair et al.,In conclusion, our results demonstrate that pseudobulk approaches lead to the best performance for the analysis of single-cell expression data based on power at equivalent type-1 error rates and on MCC for both balanced and imbalanced number of cells, from this simulated dataset.Further information on research design is available in the Supplementary InformationReporting Summary