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The concept of the histone code posits that histone modifications regulate gene functions once interpreted by epigenetic readers. A well-studied case is trimethylation of lysine 4 of histone H3 (H3K4me3), which is enriched at gene promoters. However, H3K4me3 marks are not needed for the expression of most genes, suggesting extra roles, such as influencing the 3D genome architecture. Here, we highlight an intriguing analogy between the H3K4me3-dependent induction of double-strand breaks in several recombination events and the impact of this same mark on DNA incisions for the repair of bulky lesions. We propose that Su(var)3-9, Enhancer-of-zeste and Trithorax (SET)-domain methyltransferases generate H3K4me3 to guide nucleases into chromatin spaces, the favorable accessibility of which ensures that DNA break intermediates are readily processed, thereby safeguarding genome stability.

Mechanistic target of rapamycin (mTOR)-signaling is one key driver of glioblastoma (GBM), facilitating tumor growth by promoting the shift to an anti-inflammatory, pro-cancerogenic microenvironment. Even though mTOR inhibitors such as rapamycin (RAPA) have been shown to interfere with GBM disease progression, frequently chaperoned toxic drug side effects urge the need for developing alternative or supportive treatment strategies. Importantly, previous work document that taste-immune associative learning with RAPA may be utilized to induce learned pharmacological placebo responses in the immune system. Against this background, the current study aimed at investigating the potential efficacy of a taste-immune associative learning protocol with RAPA in a syngeneic GBM rat model. Following repeated pairings of a novel gustatory stimulus with injections of RAPA, learned immune-pharmacological effects could be retrieved in GBM-bearing animals when re-exposed to the gustatory stimulus together with administering 10 % amount of the initial drug dose (0.5 mg/kg). These inhibitory effects on tumor growth were accompanied by an up-regulation of central and peripheral pro-inflammatory markers, suggesting that taste-immune associative learning with RAPA promoted the development of a pro-inflammatory anti-tumor microenvironment that attenuated GBM tumor growth to an almost identical outcome as obtained after 100 % (5 mg/kg) RAPA treatment. Together, our results confirm the applicability of taste-immune associative learning with RAPA in animal disease models where mTOR overactivation is one key driver. This proof-of-concept study may also be taken as a role model for implementing learning protocols as alternative or supportive treatment strategy in clinical settings, allowing the reduction of required drug doses and side effects without losing treatment efficacy.

Fibrosarcoma (FSA) are rare soft tissue tumors that display aggressive local behavior and invasive growth leading to high rates of tumor recurrence. While the low incidence in humans hampers detailed understanding of the disease, FSA are frequent in dogs and present potential models for the human condition. However, a lack of in-depth molecular characterization of FSA and unaffected peritumoral tissue (PTT) in both species impedes the translational potential of dogs. To address this shortcoming, we characterized canine FSA and matched skeletal muscle, adipose and connective tissue using laser-capture microdissection (LCM) and LC-MS/MS in 30 formalin-fixed paraffin embedded (FFPE) specimens. Principal component analysis of 3’530 different proteins detected across all samples clearly separates the four tissues, with several targets strongly differentiating tumor from all three PTTs. 25 proteins were exclusively found in tumor tissue in ≥80% of cases. Among these, CD68 (a macrophage marker), Optineurin (OPTN), Nuclear receptor coactivator 5 (NCOA5), RAP1GDS1 (Rap1 GTPase-GDP dissociation stimulator 1) and Stromal cell derived factor 2 like 1 (SDF2L1) were present in ≥90% of FSA. Protein expression across all FSA was highly homogeneous and characterized by MYC and TP53 signaling, hyperactive EIF2 and immune-related changes as well as strongly decreased oxidative phosphorylation and oxidative lipid metabolism. Finally, we demonstrate significant molecular homology between canine FSA and human soft-tissue sarcomas, emphasizing the relevance of studying canine FSA as a model for human FSA. In conclusion, we provide the first detailed overview of proteomic changes in FSA and surrounding PTT with relevance for the human disease., Neoplasia, 35, ISSN:1522-8002, ISSN:1476-5586

Genetically modified maize DP4114 × MON 89034 × MON 87411 × DAS-40278-9 was developed by crossing to combine four single events: DP4114, MON 89034, MON 87411 and DAS-40278-9. The GMO Panel previously assessed the four single maize events and two of the subcombinations and did not identify safety concerns. No new data on the single maize events or the assessed subcombinations were identified that could lead to modification of the original conclusions on their safety. The molecular characterisation, comparative analysis (agronomic, phenotypic and compositional characteristics) and the outcome of the toxicological, allergenicity and nutritional assessment indicate that the combination of the single maize events and of the newly expressed proteins in the four-event stack maize does not give rise to food and feed safety and nutritional concerns. Therefore, no post-market monitoring of food/feed is considered necessary. In the case of accidental release of viable four-event stack maize grains into the environment, this would not raise environmental safety concerns. The GMO Panel assessed the likelihood of interactions among the single events in eight of the maize subcombinations not previously assessed and concludes that these are expected to be as safe as the single events, the previously assessed subcombinations and the four-event stack maize. The post-market environmental monitoring plan and reporting intervals are in line with the intended uses of maize DP4114 × MON 89034 × MON 87411 × DAS-40278-9. Post-market monitoring of food/feed is not considered necessary. The GMO Panel concludes that the four-event stack maize and its subcombinations are as safe as its non-GM comparator and the tested non-GM maize varieties with respect to potential effects on human and animal health and the environment.

In 2012, EFSA issued an opinion on plants developed through cisgenesis and intragenesis. With the development of New Genomic Techniques (NGTs) in the last decade, cisgenic and intragenic plants can now be obtained with the insertion of a desired sequence in a precise location of the genome. EFSA has been requested by European Commission to provide an updated scientific opinion on the safety and the risk assessment of plants developed through cisgenesis and intragenesis, in order to (i) identify potential risks, comparing them with those posed by plants obtained by conventional breeding and Established Genomic Techniques (EGTs) and (ii) to determine the applicability of current guidelines for the risk assessment of cisgenic and intragenic plants. The conclusions of the previous EFSA opinion were reviewed, taking into consideration the new guidelines and the recent literature. The GMO panel concludes that no new risks are identified in cisgenic and intragenic plants obtained with NGTs, as compared with those already considered for plants obtained with conventional breeding and EGTs. There are no new data since the publication of the 2012 EFSA opinion that would challenge the conclusions raised in that document. The conclusions of the EFSA 2012 Scientific Opinion remain valid. The EFSA GMO Panel reiterates from these conclusions that with respect to the source of DNA and the safety of the gene product, the hazards arising from the use of a related plant-derived gene by cisgenesis are similar to those from conventional plant breeding, whereas additional hazards may arise for intragenic plants. Furthermore, the EFSA GMO Panel considers that cisgenesis and intragenesis make use of the same transformation techniques as transgenesis, and therefore, with respect to the alterations to the host genome, cisgenic, intragenic and transgenic plants obtained by random insertion do not cause different hazards. Compared to that, the use of NGTs reduces the risks associated with potential unintended modifications of the host genome. Thus, fewer requirements may be needed for the assessment of cisgenic and intragenic plants obtained through NGTs, due to site-directed integration of the added genetic material. Moreover, the GMO panel concludes that the current guidelines are partially applicable and sufficient. On a case-by-case basis, a lesser amount of data might be needed for the risk assessment of cisgenic or intragenic plants obtained through NGTs.

Circular RNAs (circRNAs) are a large class of relatively stable RNA molecules that are highly expressed in animal brains. Many circRNAs have been associated with CNS disorders accompanied by an aberrant wake-sleep cycle. However, the regulation of circRNAs in brain homeostasis over daily light-dark (LD) cycles has not been characterized. Here, we aim to quantify the daily expression changes of circRNAs in physiological conditions in healthy adult animals. Using newly generated and public RNA-Seq data, we monitored circRNA expression throughout the 12:12 h LD cycle in various mouse brain regions. We identified that Cdr1as, a conserved circRNA that regulates synaptic transmission, is highly expressed in the suprachiasmatic nucleus (SCN), the master circadian pacemaker. Despite its high stability, Cdr1as has a very dynamic expression in the SCN throughout the LD cycle, as well as a significant regulation in the hippocampus following the entry into the dark phase. Computational integration of different public datasets predicted that Cdr1as is important for regulating light entrainment in the SCN. We hypothesize that the expression changes of Cdr1as in the SCN, particularly during the dark phase, are associated with light-induced phase shifts. Importantly, our work revises the current beliefs about natural circRNA stability and suggests that the time component must be considered when studying circRNA regulation.

The designer receptor exclusively activated by designer drugs (DREADD) system is one of the most widely used chemogenetic techniques to modulate the activity of cell populations in the brains of behaving animals. DREADDs are activated by acute or chronic administration of their ligand, clozapine-N-oxide (CNO). There is, however, a current lack of a non-invasive CNO administration technique that can control for drug timing and dosing without inducing substantial distress for the animals. Here, we evaluated whether the recently developed micropipette-guided drug administration (MDA) method, which has been used as a non-invasive and minimally stressful alternative to oral gavages, may be applied to administer CNO orally to activate DREADDs in a dosing- and timing-controlled manner. Unlike standard intraperitoneal injections, administration of vehicle substances via MDA did not elevate plasma levels of the major stress hormone, corticosterone, and did not attenuate exploratory activity in the open field test. At the same time, however, administration of CNO via MDA or intraperitoneally was equally efficient in activating hM3DGq-expressing neurons in the medial prefrontal cortex, as evident by time-dependent increases in mRNA levels of neuronal immediate early genes (cFos, Arc and Zif268) and cFos-immunoreactive neurons. Compared to vehicle given via MDA, oral administration of CNO via MDA was also found to potently increase locomotor activity in mice that express hM3DGq in prefrontal neurons. Taken together, our study confirms the effectiveness of CNO given orally via MDA and provides a novel method for non-stressful, yet well controllable CNO treatments in mouse DREADD systems.

Highlights • Laser-capture microdissection (LCM) followed by RNAseq reveals detailed insights into COSCC and molecular homologies to HNSCC. • Identification of CDK4/6 as therapeutic vulnerability in COSCC. • This underlines the potential of spontaneous COSCC as a model for HNSCC to interrogate therapeutic vulnerabilities and support translation of novel therapies from bench to bedside., Spontaneously occurring canine oral squamous cell carcinomas (COSCC) are viewed as a useful model for human head and neck squamous cell carcinomas (HNSCC). To date however, the molecular basis of COSCC remains poorly understood. To identify changes pertinent to cancer cells in COSCC, we specifically analyzed tumor cells and matched normal epithelium from clinical formalin-fixed paraffin-embedded specimens using laser-capture-microdissection coupled with RNA-sequencing (RNAseq). Our results identify strong contributions of epithelial-to-mesenchymal transition (EMT), classical tumor-promoting (such as E2F, KRAS, MYC, mTORC1, and TGFB1 signaling) and immune-related pathways in the tumor epithelium of COSCC. Comparative analyses of COSCC with 43 paired tumor/normal HNSCC from The Cancer Genome Atlas revealed a high homology in transcriptional reprogramming, and identified processes associated with cell cycle progression, immune processes, and loss of cellular differentiation as likely central drivers of the disease. Similar to HNSCC, our analyses suggested a ZEB2-driven partial EMT in COSCC and identified selective upregulation of KRT14 and KRT17 in COSCC. Beyond homology in transcriptional signatures, we also found therapeutic vulnerabilities strongly conserved between the species: these included increased expression of PD-L1 and CTLA-4, coinciding with EMT and revealing the potential for immune checkpoint therapies, and overexpression of CDK4/6 that sensitized COSCC to treatment with palbociclib. In summary, our data significantly extend the current knowledge of molecular aberrations in COSCC and underline the potential of spontaneous COSCC as a model for HNSCC to interrogate therapeutic vulnerabilities and support translation of novel therapies from bench to bedside.

Infectious or noninfectious maternal immune activation (MIA) is an environmental risk factor for psychiatric and neurological disorders with neurodevelopmental etiologies. Whilst there is increasing evidence for significant health consequences, the effects of MIA on the offspring appear to be variable. Here, we aimed to identify and characterize subgroups of isogenic mouse offspring exposed to identical MIA, which was induced in C57BL6/N mice by administration of the viral mimetic, poly(I:C), on gestation day 12. Cluster analysis of behavioral data obtained from a first cohort containing >150 MIA and control offspring revealed that MIA offspring could be stratified into distinct subgroups that were characterized by the presence or absence of multiple behavioral dysfunctions. The two subgroups also differed in terms of their transcriptional profiles in cortical and subcortical brain regions and brain networks of structural covariance, as measured by ex vivo structural magnetic resonance imaging (MRI). In a second, independent cohort containing 50 MIA and control offspring, we identified a subgroup of MIA offspring that displayed elevated peripheral production of innate inflammatory cytokines, including IL-1β, IL-6, and TNF-α, in adulthood. This subgroup also showed significant impairments in social approach behavior and sensorimotor gating, whereas MIA offspring with a low inflammatory cytokine status did not. Taken together, our results highlight the existence of subgroups of MIA-exposed offspring that show dissociable behavioral, transcriptional, brain network, and immunological profiles even under conditions of genetic homogeneity. These data have relevance for advancing our understanding of the variable neurodevelopmental effects induced by MIA and for biomarker-guided approaches in preclinical psychiatric research.

In a previous scientific opinion on application EFSA-GMO-BE-2016-138, the EFSA Panel on Genetically Modified Organisms (GMO Panel) could not conclude on the comparative analysis and on the food/feed safety assessment of genetically modified (GM) oilseed rape (OSR) MS11 because of the lack of an appropriate compositional data set. Following a request from the European Commission, the GMO Panel assessed additional information related to OSR MS11 to complement the original scientific opinion. The GMO Panel concluded that the information submitted (on the composition of the two-event stack MS11 × RF3) could not be used for the assessment of the composition of OSR MS11 and requested the applicant to perform a complementary set of field trials to generate additional data. The applicant did not perform the requested field trials and did not provide any new experimental data on the composition of OSR MS11. Hence, the GMO Panel is still not in the position to conclude on either the compositional analysis or the toxicological, allergenicity or nutritional assessment of OSR MS11. Therefore, the previous conclusions of the GMO Panel still hold.

In vitro maturation (IVM) of oocytes has still a negative impact on the developmental competence of oocytes. Therefore, this study analysed the cumulus proteome of individual cumulus-oocyte complexes (COCs) with and without maturational competence, matured under in vivo or in vitro conditions (n = 5 per group). A novel, ultrasensitive mass spectrometry (MS) based protein profiling approach, using label-free quantification, was applied. The detected cumulus proteome included 2226 quantifiable proteins and was highly influenced by the maturation condition (479 differentially expressed proteins) as well as maturational competence of the corresponding oocyte (424 differentially expressed proteins). Enrichment analysis showed an overrepresentation of the complement and coagulation cascades (CCC), ECM-receptor interaction and steroid biosynthesis in cumulus of COCs that matured successfully under in vivo conditions. Verification of the origin of CCC proteins was achieved through detection of C3 secretion into the maturation medium, with significantly increasing concentrations from 12 (48.4 ng/ml) to 24 hours (68 ng/ml: p 100x higher. In summary, this study identified important pathways that are impaired in IVM cumulus, as well as potential markers of the maturational competence of oocytes., Scientific Reports, 10 (1), ISSN:2045-2322

While cancer-associated stroma (CAS) in malignant tumours is well described, stromal changes in benign forms of naturally occurring tumours remain poorly characterized. Spontaneous canine mammary carcinomas (mCA) are viewed as excellent models of human mCA. We have recently reported highly conserved stromal reprogramming between canine and human mCA based on transcriptome analysis of laser-capture-microdissected FFPE specimen. To identify stromal changes between benign and malignant mammary tumours, we have analysed matched normal and adenoma-associated stroma (AAS) from 13 canine mammary adenomas and compared them to previous data from 15 canine mCA. Our analyses reveal distinct stromal reprogramming even in small benign tumours. While similarities between AAS and CAS exist, the stromal signature clearly distinguished adenomas from mCA. The distinction between AAS and CAS is further substantiated by differential enrichment in several hallmark signalling pathways as well as differential abundance in cellular composition. Finally, we identify COL11A1, VIT, CD74, HLA-DRA, STRA6, IGFBP4, PIGR, and TNIP1 as strongly discriminatory stromal genes between adenoma and mCA, and demonstrate their prognostic value for human breast cancer. Given the relevance of canine CAS as a model for the human disease, our approach identifies disease-modulating stromal components with implications for both human and canine breast cancer.

Prenatal exposure to infectious or inflammatory insults is increasingly recognized in the etiology of neuropsychiatric diseases, including schizophrenia, autism, depression and bipolar disorder. New discoveries highlight that maternal immune activation can lead to pathological effects on brain and behavior in multiple generations. This review describes the transgenerational consequences of maternal immune activation in shaping brain and behavior anomalies and disease risk across generations. We discuss potential underlying mechanisms of transmission, by which prenatal immune activation can mediate generation-spanning changes in brain development and functions and how external influences could further determine the specificity of the phenotype across generations. The identification of the underlying mechanisms appears relevant to infection-related neuropsychiatric illnesses independently of existing diagnostic classifications and may help identifying complex patterns of generation-spanning transmission beyond genetic inheritance. The herein described principles emphasize the importance of considering ancestral infectious histories in clinical research aiming at developing new preventive treatment strategies against infection-related neurodevelopmental disorders and mental illnesses.

Editorial on the Research Topic. Looking at the Complete Picture: Tackling Broader Factors Important for Advancing the Validity of Preclinical Models in Disease, Frontiers in Behavioral Neuroscience, 16, ISSN:1662-5153

Background Neuropsychiatric disorders, such as schizophrenia (SZ) and autism spectrum disorder (ASD), are common, multi-factorial and multi-symptomatic disorders. Ample evidence implicates oxidative stress, deficient repair of oxidative DNA lesions and DNA damage in the development of these disorders. However, it remains unclear whether insufficient DNA repair and resulting DNA damage are causally connected to their aetiopathology, or if increased levels of DNA damage observed in patient tissues merely accumulate as a consequence of cellular dysfunction. To assess a potential causal role for deficient DNA repair in the development of these disorders, we behaviourally characterized a mouse model in which CaMKIIa-Cre-driven postnatal conditional knockout (KO) of the core base-excision repair (BER) protein XRCC1 leads to accumulation of unrepaired DNA damage in the forebrain. Results CaMKIIa-Cre expression caused specific deletion of XRCC1 in the dorsal dentate gyrus (DG), CA1 and CA2 and the amygdala and led to increased DNA damage therein. While motor coordination, cognition and social behaviour remained unchanged, XRCC1 KO in the forebrain caused increased anxiety-like behaviour in males, but not females, as assessed by the light–dark box and open field tests. Conversely, in females but not males, XRCC1 KO caused an increase in learned fear-related behaviour in a cued (Pavlovian) fear conditioning test and a contextual fear extinction test. The relative density of the GABA(A) receptor alpha 5 subunit (GABRA5) was reduced in the amygdala and the dorsal CA1 in XRCC1 KO females, whereas male XRCC1 KO animals exhibited a significant reduction of GABRA5 density in the CA3. Finally, assessment of fast-spiking, parvalbumin-positive (PV) GABAergic interneurons revealed a significant increase in the density of PV+ cells in the DG of male XRCC1 KO mice, while females remained unchanged. Conclusions Our results suggest that accumulation of unrepaired DNA damage in the forebrain alters the GABAergic neurotransmitter system and causes behavioural deficits in relation to innate and learned anxiety in a sex-dependent manner. Moreover, the data uncover a previously unappreciated connection between BER deficiency, unrepaired DNA damage in the hippocampus and a sex-specific anxiety-like phenotype with implications for the aetiology and therapy of neuropsychiatric disorders., BMC Biology, 20, ISSN:1741-7007

EFSA was asked by the european Commission to develop criteria as advice for consideration for the risk assessment of plants produced by targeted mutagenesis, cisgenesis and intragenesis. EFSA proposes in this statement six main criteria to assist the risk assessment of these plants. The first four criteria are related to the molecular characterisation of the genetic modification introduced in the recipient plant. The four criteria evaluate whether any exogenous DNA sequence(s) is/are present (Criterion 1), whether such sequence derives from the breeders' gene pool (Criterion 2), the type of integration (Criterion 3) and whether any endogenous plant gene is interrupted (Criterion 4). Depending on the evaluation of the above criteria, the product can be a genome edited plant where no exogenous DNA sequence is present, or a cisgenic or intragenic plant where the cisgenic and intragenic sequence are introduced by targeted insertion and no plant endogenous genes are interrupted. In these cases, two more criteria are assessed to evaluate the history of safe use (Criterion 5) and the structure and function of the new allele (Criterion 6). If cisgenic and intragenic sequence are introduced by random integration without interruption of an endogenous gene, or when no risk is identified when an endogenous gene is interrupted, the criteria 5 and 6 will also be assessed. Evaluating the history of safe use is an important part of the proportionate risk assessment of cisgenic, intragenic and genome-edited plants since the newly introduced allele may already be present in nature. However, when the history of safe use cannot be sufficiently demonstrated, the function and structure of the introduced allele should be carefully assessed. Recommendations are also included on the aspects that need further elaboration for full applicability of the criteria proposed herein are also included.

Prenatal exposure to infectious or noninfectious immune activation is an environmental risk factor for neurodevelopmental disorders and mental illnesses. Recent research using animal models suggests that maternal immune activation (MIA) during early to middle stages of pregnancy can induce transgenerational effects on brain and behavior, likely via inducing stable epigenetic modifications across generations. Using a mouse model of viral-like MIA, which is based on gestational treatment with poly(I:C), the present study explored whether transgenerational effects can also emerge when MIA occurs in late pregnancy. Our findings demonstrate that the direct descendants born to poly(I:C)-treated mothers display deficits in temporal order memory, which are similarly present in second- and third-generation offspring. These transgenerational effects were mediated via both the maternal and paternal lineages and were accompanied by transient changes in maternal care. In addition to the cognitive effects, late prenatal immune activation induced generation-spanning effects on the prefrontal expression of gamma-aminobutyric acid (GABA)ergic genes, including parvalbumin and distinct alpha-subunits of the GABAA receptor. Together, our results suggest that MIA in late pregnancy has the potential to affect cognitive functions and prefrontal gene expression patterns in multiple generations, highlighting its role in shaping disease risk across generations.

Much confusion has been generated in the safety assessment of food-grade TiO2 (E171) by the comingling of studies conducted on submicron-sized particles with those examining the toxicity of more minuscule counterparts. As E171 displays a nano-sized tail in its particle distribution (up to 36 % of particles with a diameter < 100 nm), it was thought that potential hazards of this food additive can be extrapolated from studies on thoroughly nanoscale formulations. This simplistic procedure may, however, overestimate the effects of the nano-sized tail of E171 because TiO2 particles readily aggregate or agglomerate in aqueous suspensions and biological matrices. The resulting larger clusters display a reduced oral bioavailability in comparison to the same material in nano-sized dimensions. Also, even if taken up in trace amounts, the smaller particles likely remain appended to larger particles or clusters and these aggregates or conglomerates may nullify to a great extent their ���nano��� characteristics. The purpose of this review is, therefore, to reevaluate the literature on the toxicity of TiO2 particles focusing on studies that are directly relevant for the assessment of E171. The purpose is not to avert a ban on the use of E171 in food, which might well be justified in light of the uncertainties associated with this additive employed solely for its colorant properties. Instead, it will be important to avoid in the future this same bias towards a fictional ���nano��� hazard, especially when evaluating more innovative engineered particles that confer true benefits for example by enhancing nutritional properties, quality, freshness, traceability or sustainability of food., EXCLI Journal; 21:Doc279; ISSN 1611-2156

Synthetic biology (SynBio) is an interdisciplinary field at the interface of molecular engineering and biology aiming to develop new biological systems and impart new functions to living cells, tissues and organisms. EFSA has been asked by the European Commission to evaluate SynBio developments in agri-food with the aim of identifying the adequacy and sufficiency of existing guidelines for risk assessment and determine if updated guidance is needed. In this context, the GMO Panel has previously adopted an Opinion evaluating the SynBio developments in agri-food/feed and the adequacy and sufficiency of existing guidelines for the molecular characterisation and environmental risk assessment of genetically modified plants (GMPs) obtained through SynBio and reaching the market in the next decade. Complementing the above, in this Opinion, the GMO Panel evaluated the adequacy and sufficiency of existing guidelines for the food and feed risk assessment of GMPs obtained through SynBio. Using selected hypothetical case studies, the GMO Panel did not identify novel potential hazards and risks that could be posed by food and feed from GMPs obtained through current and near future SynBio approaches; considers that the existing guidelines are adequate and sufficient in some Synbio applications; in other cases, existing guidelines may be just adequate and hence need updating; areas needing updating include those related to the safety assessment of new proteins and the comparative analysis. The GMO Panel recommends that future guidance documents provide indications on how to integrate the knowledge available from the SynBio design and modelling in the food and feed risk assessment and encourages due consideration to be given to food and feed safety aspects throughout the SynBio design process as a way to facilitate the risk assessment of SynBio GMPs and reduce the amount of data required.

Background DNA-crosslinking agents like cisplatin and mitomycin C (MMC) are indispensible for the treatment of many solid malignancies. These anticancer drugs generate DNA interstrand crosslinks (ICLs) that cause cell death by blocking replication forks. Many factors counteracting ICL-induced DNA replication stress, including the Fanconi anemia (FA) pathway, are regulated by ubiquitination and, therefore, ubiquitin ligases are potential targets for the sensitization of cancer cells to crosslinking agents. In this study, we investigated the function of the CRL4 ubiquitin ligase in modulating the response of cancer cells to ICL induction. Methods The two cullin paralogs CUL4A and CUL4B, which form the CRL4 ligase scaffold, were depleted in cancer cells by small interfering RNA followed by analysis of the cellular and biochemical responses to ICLs elicited upon cisplatin or MMC treatment. Results We report that the combined depletion of CUL4A and CUL4B weakens an FA pathway-dependent S phase checkpoint response. CRL4 positively stimulates the monoubiquitination of FANCD2 required for the recruitment of XPF-ERCC1, a structure-specific endonuclease that, in turn, contributes to the display of single-stranded DNA (ssDNA) at ICLs. After CRL4 down regulation, the missing ssDNA results in reduced recruitment of RPA, thereby dampening activation of ATR and CHK1 checkpoint kinases and allowing for S phase progression despite ICL induction. Conclusion Our findings indicate that CRL4 promotes cell survival by potentiating an FA pathway-dependent ssDNA-RPA signaling platform installed at ICLs. The anticancer efficacy of crosslinking agents may, therefore, be enhanced by down regulating CRL4 activity.

This guidance document provides harmonised and flexible methodologies to apply scientific criteria and prioritisation methods for grouping chemicals into assessment groups for human risk assessment of combined exposure to multiple chemicals. In the context of EFSA’s risk assessments, the problem formulation step defines the chemicals to be assessed in the terms of reference usually through regulatory criteria often set by risk managers based on legislative requirements. Scientific criteria such as hazard‐driven criteria can be used to group these chemicals into assessment groups. In this guidance document, a framework is proposed to apply hazard‐driven criteria for grouping of chemicals into assessment groups using mechanistic information on toxicity as the gold standard where available (i.e. common mode of action or adverse outcome pathway) through a structured weight of evidence approach. However, when such mechanistic data are not available, grouping may be performed using a common adverse outcome. Toxicokinetic data can also be useful for grouping, particularly when metabolism information is available for a class of compounds and common toxicologically relevant metabolites are shared. In addition, prioritisation methods provide means to identify low‐priority chemicals and reduce the number of chemicals in an assessment group. Prioritisation methods include combined risk‐based approaches, risk‐based approaches for single chemicals and exposure‐driven approaches. Case studies have been provided to illustrate the practical application of hazard‐driven criteria and the use of prioritisation methods for grouping of chemicals in assessment groups. Recommendations for future work are discussed., This publication is linked to the following EFSA Supporting Publications article: http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2021.EN-7029/full

Maize NK603 × T25 × DAS-40278-9 (three-event stack maize) was produced by conventional crossing to combine three single events: NK603, T25 and DAS-40278-9. The GMO Panel previously assessed the three single maize events and two of the subcombinations and did not identify safety concerns. No new data on the single maize events or the two subcombinations were identified that could lead to modification of the original conclusions on their safety. The molecular characterisation, comparative analysis (agronomic, phenotypic and compositional characteristics) and the outcome of the toxicological, allergenicity and nutritional assessment indicate that the combination of the single maize events and of the newly expressed proteins in the three-event stack maize does not give rise to food and feed safety and nutritional concerns. The GMO Panel concludes that the three-event stack maize, as described in this application, is as safe as the non-GM comparator and the selected non-GM reference varieties. In the case of accidental release of viable grains of the three-event stack maize into the environment, this would not raise environmental safety concerns. The GMO Panel assessed the likelihood of interactions among the single events in one of the maize subcombinations not previously assessed and concludes that these are expected to be as safe as the single events, the previously assessed subcombinations and the three-event stack maize. The post-market environmental monitoring plan and reporting intervals are in line with the intended uses of the three-event stack maize. Post-market monitoring of food/feed is not considered necessary. The GMO Panel concludes that the three-event stack maize and its subcombinations are as safe as the non-GM comparator and the selected non-GM reference varieties with respect to potential effects on human and animal health and the environment., European Commission: EFSA-Q-2019-00808.

Mutations are changes in the genetic material that may be transmitted to subsequent generations. Mutations appear spontaneously in nature and are one of the underlying driving forces of evolution. In plants, in vivo and in vitro random mutagenesis relies on the application of physical and chemical mutagens to increase the frequency of mutations thus accelerating the selection of varieties with important agronomic traits. The European Commission has requested EFSA to provide a more detailed description of in vivo and in vitro random mutagenesis techniques and the types of mutations and mechanisms involved, to be able to conclude on whether in vivo and in vitro random mutagenesis techniques are to be considered different techniques. To address the European Commission request, a literature search was conducted to collect information on the random mutagenesis techniques used in plants both in vivo and in vitro, on the type of mutations generated by such techniques and on the molecular mechanisms underlying formation of those mutations. The GMO Panel concludes that most physical and chemical mutagenesis techniques have been applied both in vivo and in vitro; the mutation process and the repair mechanisms act at cellular level and thus there is no difference between application of the mutagen in vivo or in vitro; and the type of mutations induced by a specific mutagen are expected to be the same, regardless of whether such mutagen is applied in vivo or in vitro. Indeed, the same mutation and the derived trait in a given plant species can be potentially obtained using both in vivo and in vitro random mutagenesis and the resulting mutants would be indistinguishable. Therefore, the GMO Panel concludes that the distinction between plants obtained by in vitro or in vivo approaches is not justified., European Commission: EFSA-Q-2020-00445

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer worldwide. Cancer-associated stroma (CAS) is central to tumor development and strongly influences therapy response. Perineural infiltration (PNI) represents a major risk factor for cSCC and likely influences CAS reprogramming. However, stromal reprogramming in cSCC remains poorly characterized, and it is unknown whether and how PNI influences CAS. To address these questions, we analyzed CAS and matched normal stroma from 20 cSCC cases (11 without PNI and 9 with PNI) by laser-capture microdissection using RNA sequencing. Our analysis reveals extensive stromal reprogramming strongly driven by changes in immune cells, as validated using immunohistochemistry. Furthermore, CAS of cSCC displays markers of immune exhaustion, and multiplex spatial analysis suggests that PD-L1 expression on NK T cells contributes to T-cell exhaustion and immunosuppression. Finally, PNI is characterized by increased IL-17A. In PNI-negative cases, IL-17A derives predominantly from CD3+ cells. However, with PNI, we observe an increased contribution of fibroblasts to high IL-17A, which coincides with a significant increase in FAP+ cells. Our analysis elucidates the molecular landscape of CAS in cSCC and identifies the presence of immunosuppressive mechanisms, supporting further research into immunotherapy and anti‒IL-17A in cSCC, especially for cases with PNI.

Human endogenous retroviruses (HERVs) are remnants of infections that took place several million years ago and represent around 8% of the human genome. Despite evidence implicating increased expression of HERV type W envelope (HERV-W ENV) in schizophrenia and bipolar disorder, it remains unknown whether such expression is associated with distinct clinical or biological characteristics and symptoms. Accordingly, we performed unsupervised two-step clustering of a multivariate data set that included HERV-W ENV protein antigenemia, serum cytokine levels, childhood trauma scores, and clinical data of cohorts of patients with schizophrenia (n = 29), bipolar disorder (n = 43) and healthy controls (n = 32). We found that subsets of patients with schizophrenia (~41%) and bipolar disorder (~28%) show positive antigenemia for HERV-W ENV protein, whereas the large majority (96%) of controls was found to be negative for ENV protein. Unsupervised cluster analysis identified the presence of two main clusters of patients, which were best predicted by the presence or absence of HERV-W ENV protein. HERV-W expression was associated with increased serum levels of inflammatory cytokines and higher childhood maltreatment scores. Furthermore, patients with schizophrenia who were positive for HERV-W ENV protein showed more manic symptoms and higher daily chlorpromazine (CPZ) equivalents, whereas HERV-W ENV positive patients with bipolar disorder were found to have an earlier disease onset than those who were negative for HERV-W ENV protein. Taken together, our study suggest that HERV-W ENV protein antigenemia and cytokines can be used to stratify patients with major mood and psychotic disorders into subgroups with differing inflammatory and clinical profiles.

The EFSA has updated the Guidance on risk assessment of the application of nanoscience and nanotechnologies in the food and feed chain, human and animal health. It covers the application areas within EFSA’s remit, including novel foods, food contact materials, food/feed additives and pesticides. The updated guidance, now Scientific Committee Guidance on nano risk assessment (SC Guidance on Nano‐RA), has taken account of relevant scientific studies that provide insights to physico‐chemical properties, exposure assessment and hazard characterisation of nanomaterials and areas of applicability. Together with the accompanying Guidance on Technical requirements for regulated food and feed product applications to establish the presence of small particles including nanoparticles (Guidance on Particle‐TR), the SC Guidance on Nano‐RA specifically elaborates on physico‐chemical characterisation, key parameters that should be measured, methods and techniques that can be used for characterisation of nanomaterials and their determination in complex matrices. The SC Guidance on Nano‐RA also details aspects relating to exposure assessment and hazard identification and characterisation. In particular, nanospecific considerations relating to in vitro/in vivo toxicological studies are discussed and a tiered framework for toxicological testing is outlined. Furthermore, in vitro degradation, toxicokinetics, genotoxicity, local and systemic toxicity as well as general issues relating to testing of nanomaterials are described. Depending on the initial tier results, additional studies may be needed to investigate reproductive and developmental toxicity, chronic toxicity and carcinogenicity, immunotoxicity and allergenicity, neurotoxicity, effects on gut microbiome and endocrine activity. The possible use of read‐across to fill data gaps as well as the potential use of integrated testing strategies and the knowledge of modes or mechanisms of action are also discussed. The Guidance proposes approaches to risk characterisation and uncertainty analysis., This publication is linked to the following EFSA Journal article: http://onlinelibrary.wiley.com/doi/10.2903/j.efsa.2021.6769/full This publication is linked to the following EFSA Supporting Publications article: http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2021.EN-6804/full

Following a mandate from the European Commission, EFSA has developed a Guidance on Technical Requirements (Guidance on Particle‐TR), defining the criteria for assessing the presence of a fraction of small particles, and setting out information requirements for applications in the regulated food and feed product areas (e.g. novel food, food/feed additives, food contact materials and pesticides). These requirements apply to particles requiring specific assessment at the nanoscale in conventional materials that do not meet the definition of engineered nanomaterial as set out in the Novel Food Regulation (EU) 2015/2283. The guidance outlines appraisal criteria grouped in three sections, to confirm whether or not the conventional risk assessment should be complemented with nanospecific considerations. The first group addresses solubility and dissolution rate as key physicochemical properties to assess whether consumers will be exposed to particles. The second group establishes the information requirements for assessing whether the conventional material contains a fraction or consists of small particles, and its characterisation. The third group describes the information to be presented for existing safety studies to demonstrate that the fraction of small particles, including particles at the nanoscale, has been properly evaluated. In addition, in order to guide the appraisal of existing safety studies, recommendations for closing the data gaps while minimising the need for conducting new animal studies are provided. This Guidance on Particle‐TR complements the Guidance on risk assessment of nanomaterials to be applied in the food and feed chain, human and animal health updated by the EFSA Scientific Committee as co‐published with this Guidance. Applicants are advised to consult both guidance documents before conducting new studies., This publication is linked to the following EFSA Journal article: http://onlinelibrary.wiley.com/doi/10.2903/j.efsa.2021.6768/full This publication is linked to the following EFSA Supporting Publications article: http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2021.EN-6804/full

The EFSA Scientific Committee was asked to provide guidance on the most appropriate in vivo tests to follow up on positive in vitro results for aneugenicity, and on the approach to risk assessment for substances that are aneugenic but not clastogenic nor causing gene mutations. The Scientific Committee confirmed that the preferred approach is to perform an in vivo mammalian erythrocyte micronucleus test with a relevant route of administration. If this is positive, it demonstrates that the substance is aneugenic in vivo. A negative result with evidence that the bone marrow is exposed to the test substance supports a conclusion that aneugenic activity is not expressed in vivo. If there is no evidence of exposure to the bone marrow, a negative result is viewed as inconclusive and further studies are required. The liver micronucleus assay, even though not yet fully validated, can provide supporting information for substances that are aneugenic following metabolic activation. The gastrointestinal micronucleus test, conversely, to be further developed, may help to assess aneugenic potential at the initial site of contact for substances that are aneugenic in vitro without metabolic activation. Based on the evidence in relation to mechanisms of aneugenicity, the Scientific Committee concluded that, in principle, health‐based guidance values can be established for substances that are aneugenic but not clastogenic nor causing gene mutations, provided that a comprehensive toxicological database is available. For situations in which the toxicological database is not sufficient to establish health‐based guidance values, some approaches to risk assessment are proposed. The Scientific Committee recommends further development of the gastrointestinal micronucleus test, and research to improve the understanding of aneugenicity to support risk assessment., This publication is linked to the following EFSA Supporting Publications article: http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2021.EN-6814/full