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Mitochondrial base editing tools hold great promise for the investigation and treatment of mitochondrial diseases. Mitochondrial DNA base editors (mitoBEs) integrate a programmable transcription-activator-like effector (TALE) protein with single-stranded DNA deaminase (TadA8e-V106W, APOBEC1, etc.) and nickase (MutH, Nt.BspD6I(C), etc.) to achieve heightened precision and efficiency in mitochondrial base editing. This innovative mitochondrial base editing tool exhibits a number of advantages, including strand-selectivity for editing, high efficiency, and the capacity to perform diverse types of base editing on the mitochondrial genome by employing various deaminases. In this context, we provide a detailed experimental protocol for mitoBEs to assist others in achieving proficient mitochondrial base editing.

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Pro-B- and pre-B-cells are consecutive entities in early B-cell development, representing cells of origin for B-cell precursor acute lymphoid leukemia (BCP-ALL). Normal B-cell differentiation is critically regulated by specific transcription factors (TFs). Accordingly, TF-encoding genes are frequently deregulated or mutated in BCP-ALL. Recently, we described TF-codes which delineate physiological activities of selected groups of TF-encoding genes in hematopoiesis including B-cell development. Here, we exploited these codes to uncover regulatory connections between particular TFs in pro-B- and pre-B-cells via an analysis of developmental TFs encoded by NKL and TALE homeobox genes and by ETS and T-box genes. Comprehensive expression analyses in BCP-ALL cell lines helped identify validated models to study their mutual regulation in vitro. Knockdown and overexpression experiments and subsequent RNA quantification of TF-encoding genes in selected model cell lines revealed activating, inhibitory or absent connections between nine TFs operating in early B-cell development, including HLX, MSX1, IRX1, MEIS1, ETS2, ERG, SPIB, EOMES, and TBX21. In addition, genomic profiling revealed BCP-ALL subtype-specific copy number alterations of ERG at 21q22, while a deletion of the TGFbeta-receptor gene TGFBR2 at 3p24 resulted in an upregulation of EOMES. Finally, we combined the data to uncover gene regulatory networks which control normal differentiation of early B-cells, collectively endorsing more detailed evaluation of BCP-ALL subtypes.

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Fetal death is defined as the spontaneous cessation of cardiac activity after fourteen weeks of amenorrhea. In France, the prevalence of fetal death after 22 weeks is between 3.2 and 4.4/1000 births. Regarding the prevention of fetal death in the general population, it is not recommended to counsel for rest and not to prescribe vitamin A, vitamin D nor micronutrient supplementation for the sole purpose of reducing the risk of fetal death (Weak recommendations; Low quality of evidence). It is not recommended to prescribe aspirin (Weak recommendation; Very low quality of evidence). It is recommended to offer vaccination against influenza in epidemic periods and against SARS-CoV-2 (Strong recommendations; Low quality of evidence). It is not recommended to systematically look for nuchal cord encirclements during prenatal screening ultrasounds (Strong Recommendation; Low Quality of Evidence) and not to perform systematic antepartum monitoring by cardiotocography (Weak Recommendation; Very Low Quality of Evidence). It is not recommended to ask women to perform an active fetal movement count to reduce the risk of fetal death (Strong Recommendation; High Quality of Evidence). Regarding evaluation in the event of fetal death, it is suggested that an external fetal examination be systematically offered (Expert opinion). It is recommended that a fetopathological and anatomopathological examination of the placenta be carried out to participate in cause identification (Strong Recommendation. Moderate quality of evidence). It is recommended that chromosomal analysis by microarray testing be performed rather than conventional karyotype, in order to be able to identify a potentially causal anomaly more frequently (Strong Recommendation, moderate quality of evidence); to this end, it is suggested that postnatal sampling of the placental fetal surface for genetic purposes be preferred (Expert Opinion). It is suggested to test for antiphospholipid antibodies and systematically perform a Kleihauer test and a test for irregular agglutinins (Expert opinion). It is suggested to offer a summary consultation, with the aim of assessing the physical and psychological status of the parents, reporting the results, discussing the cause and providing information on monitoring for a subsequent pregnancy (Expert opinion). Regarding announcement and support, it is suggested to announce fetal death without ambiguity, using simple words and adapting to each situation, and then to support couples with empathy in the various stages of their care (Expert opinion). Regarding management, it is suggested that, in the absence of a situation at risk of disseminated intravascular coagulation or maternal vitality, the patient's wishes should be taken into account when determining the time between the diagnosis of fetal death and induction of birth. Returning home is possible if it's the patient wish (Expert opinion). In all situations excluding maternal life-threatening emergencies, the preferred mode of delivery is vaginal delivery, regardless the history of cesarean section(s) history (Expert opinion). In the event of fetal death, it is recommended that mifepristone 200mg be prescribed at least 24hours before induction, to reduce the delay between induction and delivery (Low recommendation. Low quality of evidence). There are insufficient data in the literature to make a recommendation regarding the route of administration (vaginal or oral) of misoprostol, neither the type of prostaglandin to reduce induction-delivery time or maternal morbidity. It is suggested that perimedullary analgesia be introduced at the start of induction if the patient asks, regardless of gestational age. It is suggested to prescribe cabergoline immediately in the postpartum period in order to avoid lactation, whatever the gestational age, after discussing the side effects of the treatment with the patient (Expert opinion). The risk of recurrence of fetal death after unexplained fetal death does not appear to be increased in subsequent pregnancies, and data from the literature are insufficient to make a recommendation on the prescription of aspirin. In the event of a history of fetal death due to vascular issues, low-dose aspirin is recommended to reduce perinatal morbidity, and should not be combined with heparin therapy (Low recommendation, very low quality of evidence). It is suggested not to recommend an optimal delay before initiating another pregnancy just because of the history of fetal death. It is suggested that the woman and co-parent be informed of the possibility of psychological support. Fetal heart rate monitoring is not indicated solely because of a history of fetal death. It is suggested that delivery not be systematically induced. However, induction can be considered depending on the context and parental request. The gestational age will be discussed, taking into account the benefits and risks, especially before 39 weeks. If a cause of fetal death is identified, management will be adapted on a case-by-case basis (expert opinion). In the event of fetal death occurring in a twin pregnancy, it is suggested that the surviving twin be evaluated as soon as the diagnosis of fetal death is made. In the case of dichorionic pregnancy, it is suggested to offer ultrasound monitoring on a monthly basis. It is suggested not to deliver prematurely following fetal death of a twin. If fetal death occurs in a monochorionic twin pregnancy, it is suggested to contact the referral competence center, in order to urgently look for signs of acute fetal anemia on ultrasound in the surviving twin, and to carry out weekly ultrasound monitoring for the first month. It is suggested not to induce birth immediately.

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PBX1 is a transcription factor that can promote the occurrence of various tumors and play a reg-ulatory role in tumor growth, metastasis, invasion, and drug resistance. Furthermore, a variant generated by fusion of E2A and PBX1, E2A-PBX1, has been found in 25% of patients with childhood acute lymphoblastic leukemia. Thus, PBX1 is a potential therapeutic target for many cancers. Here, we describe the structure of PBX1 and E2A-PBX1 as well as the molecular mecha-nisms whereby these proteins promote tumorigenesis to provide future research directions for developing new treatments. We show that PBX1 and E2A-PBX1 induce the development of highly malignant and difficult-to-treat solid and blood tumors. The development of specific drugs against their targets may be a good therapeutic strategy for PBX1-related cancers. Furthermore, we strongly recommend E2A-PBX1 as one of the genes for prenatal screening to reduce the incidence of childhood hematological malignancies.

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Bacterial leaf streak (BLS), caused by Xanthomonas oryzae pv. oryzicola (Xoc), is a major bacterial disease in rice. Transcription activator-like effectors (TALEs) from Xanthomonas can induce host susceptibility (S) genes and facilitate infection. However, knowledge of the function of Xoc TALEs in promoting bacterial virulence is limited. In this study, we demonstrated the importance of Tal10a for the full virulence of Xoc. Through computational prediction and gene expression analysis, we identified the hexokinase gene OsHXK5 as a host target of Tal10a. Tal10a directly binds to the gene promoter region and activates the expression of OsHXK5. CRISPR/Cas9-mediated gene editing in the effector binding element (EBE) of OsHXK5 significantly increases rice resistance to Xoc, while OsHXK5 overexpression enhances the susceptibility of rice plants and impairs rice defense responses. Moreover, simultaneous editing of the promoters of OsSULTR3;6 and OsHXK5 confers robust resistance to Xoc in rice. Taken together, our findings highlight the role of Tal10a in targeting OsHXK5 to promote infection and suggest that OsHXK5 represents a potential target for engineering rice resistance to Xoc.

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Glycosylase base editor (GBE) can induce C-to-G transversion in mammalian cells, showing great promise for the treatment of human genetic disorders. However, the limited efficiency of transversion and the possibility of off-target effects caused by Cas9 restrict its potential clinical applications. In our recent study, we have successfully developed TaC9-CBE and TaC9-ABE by separating nCas9 and deaminase, which eliminates the Cas9-dependent DNA off-target effects without compromising editing efficiency. We developed a novel GBE called TaC9-GBEYE1, which utilizes the deaminase and UNG-nCas9 guided by TALE and sgRNA, respectively. TaC9-GBEYE1 showed comparable levels of on-target editing efficiency to traditional GBE at 19 target sites, without any off-target effects caused by Cas9 or TALE. The TaC9-GBEYE1 is a safe tool for gene therapy.

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Three-amino-loop-extension (TALE) family belongs to the homeobox gene superfamily and occurs widely in plants, playing a crucial role in regulating their growth and development. Currently, genome-wide analysis of the TALE family has been completed in many plants. However, the systematic identification and hormone response analysis of the TALE gene family in barley are still lacking. In this study, 21 TALE candidate genes were identified in barley, which can be divided into KNOX and BELL subfamilies. Barley TALE members in the same subfamily of the phylogenetic tree have analogically conserved motifs and gene structures, and segmental duplications are largely responsible for the expansion of the HvTALE family. Analysis of TALE orthologous and homologous gene pairs indicated that the HvTALE family has mainly undergone purifying selective pressure. Through spatial structure simulation, HvKNOX5-HvKNOX6 and HvKNOX5-HvBELL11 complexes are all formed through hydrogen bonding sites on both the KNOX2 and homeodomain (HD) domains of HvKNOX5, which may be essential for protein interactions among the HvTALE family members. Expression pattern analyses reveal the potential involvement of most HvTALE genes in responses to exogenous hormones. These results will lay the foundation for regulation and function analyses of the barley TALE gene family in plant growth and development by hormone regulation.

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One of the most recent advances in the genome editing field has been the addition of "TALE Base Editors", an innovative platform for cell therapy that relies on the deamination of cytidines within double strand DNA, leading to the formation of an uracil (U) intermediate. These molecular tools are fusions of transcription activator-like effector domains (TALE) for specific DNA sequence binding, split-DddA deaminase halves that will, upon catalytic domain reconstitution, initiate the conversion of a cytosine (C) to a thymine (T), and an uracil glycosylase inhibitor (UGI). We developed a high throughput screening strategy capable to probe key editing parameters in a precisely defined genomic context in cellulo, excluding or minimizing biases arising from different microenvironmental and/or epigenetic contexts. Here we aimed to further explore how target composition and TALEB architecture will impact the editing outcomes. We demonstrated how the nature of the linker between TALE array and split DddAtox head allows us to fine tune the editing window, also controlling possible bystander activity. Furthermore, we showed that both the TALEB architecture and spacer length separating the two TALE DNA binding regions impact the target TC editing dependence by the surrounding bases, leading to more restrictive or permissive editing profiles.

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BACKGROUND: Three Amino acid Loop Extension (TALE) belongs to the homeobox group of genes that are important constituents of plant systems. The TALE gene family is instrumental not only in growth and development but also plays an essential role in regulating plant response to environmental adversaries. RESULTS: In the present study, we isolated 21 CsTALE genes from the cucumber (Cucumis sativus L.) genome database. Bioinformatics tools were put in place to understand the structural and functional components of the CsTALE gene family. The evolutionary analysis dissected them into seven subclades (KNOX-I, KNOX-II, and BELL-I to BELL-V). The cis-acting elements in the promoter region of CsTALE genes disclosed that they are key regulators of hormonal and stress-related processes. Additionally, the STRING database advocated the concerting role of CsTALE proteins with other key transcription factors potent in plant developmental biology. The CsmiR319 and CsmiR167a-3p targeting the CsTALE15 and CsTALE16, respectively, further assert the importance of the CsTALE gene family posttranscriptional-related processes. Tissue-specific gene expression unfolded the fundamental involvement of CsTALE genes as they were expressed throughout the developmental stages. Under waterlogging stress, the CsTALE17 expressed significantly higher values in WL, WL-NAA, and WL-ETH but not in WL-MeJA-treated samples. CONCLUSIONS: The present study reveals the evolution and functions of the CsTALE gene family in cucumber. Our work will provide a platform that will help future researchers address the issue of waterlogging stress in the Yangtze River Delta.

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Programmable nucleases-based genome editing systems offer several advantages, such as high editing efficiency, high product purity, and fewer editing by-products. They have been widely used in biopharmaceutical research and crop engineering. Given the diverse needs for research and application, developing functional base editors has become a major focus in the field of genome editing. Currently, genome editing systems derived from clustered regularly interspaced short palindromic repeats and CRISPR-associated (CRISPR-Cas) and transcription activator-like effector (TALE) systems include single base editors, dual base editors, mitochondrial base editors, and CRISPR-related transposase systems. This review provides a comprehensive overview of the development of base editing systems, summarizes the characteristics, off-target effects, optimization, and improvement strategies of various base editors, and provides insights for further improvement and application of genome editing systems.

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This study primarily aimed to develop a program that aids parents of children with Special Educational Needs (SEN), placing emphasis on the challenges encountered in communication and understanding. The Eidemiller and Justizkis's (2008) Family Relationship Analysis (FRA) served as the core method of the research. As inclusive education progresses, new challenges incessantly emerge necessitating immediate effective solutions. This research spotlights the triad of teachers, psychologists, and parents. A cornerstone of this research is the emphasis on nurturing a unique bond between school psychologists or teachers and parents. This bond should be anchored in mutual trust, motivation, and a conducive psychological environment. The overarching goal is to boost parental motivation, alleviate concerns linked to the child's condition, reinforce family relationships, and cultivate a positive enabling environment for the child. The study culminated in the rollout of a tailored program connecting parents with educational professionals. This program encompassed lectures and exercises delving into understanding challenges, coupled with innovative methods like fairy tale activities and initiatives to foster tolerance. Feedback indicated a notable positive impact on parents. The research underscores the necessity of fostering parental confidence and assuaging fears regarding their child's condition. In this context, the influence of peers and friends on the development of inclusive education should be considered in future studies.

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At present, there are a variety of different approaches to the targeted regulation of gene expression. However, most approaches are devoted to the activation of gene transcription, and the methods for gene silencing are much fewer in number. In this review, we describe the main systems used for the targeted suppression of gene expression (including RNA interference (RNAi), chimeric transcription factors, chimeric zinc finger proteins, transcription activator-like effectors (TALEs)-based repressors, optogenetic tools, and CRISPR/Cas-based repressors) and their application in eukaryotes-plants and animals. We consider the advantages and disadvantages of each approach, compare their effectiveness, and discuss the peculiarities of their usage in plant and animal organisms. This review will be useful for researchers in the field of gene transcription suppression and will allow them to choose the optimal method for suppressing the expression of the gene of interest depending on the research object.

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Xanthomonas oryzae pv. oryzae (Xoo) injects major transcription activator-like effectors (TALEs) into plant cells to activate susceptibility (S) genes for promoting bacterial leaf blight in rice. Numerous resistance (R) genes have been used to construct differential cultivars of rice to identify races of Xoo, but the S genes were rarely considered. Different edited lines of rice cv. Kitaake were constructed using CRISPR/Cas9 gene-editing, including single, double and triple edits in the effector-binding elements (EBEs) located in the promoters of rice S genes OsSWEET11a, OsSWEET13 and OsSWEET14. The near-isogenic lines (NILs) were used as tracers to detect major TALEs (PthXo1, PthXo2, PthXo3 and their variants) in 50 Xoo strains. The pathotypes produced on the tracers determined six major TALE types in the 50 Xoo strains. The presence of the major TALEs in Xoo strains was consistent with the expression of S genes in the tracers, and it was also by known genome sequences. The EBE editing had little effect on agronomic traits, which was conducive to balancing yield and resistance. The rice-tracers generated here provide a valuable tool to track major TALEs of Xoo in Asia which then shows what rice cultivars are needed to combat Xoo in the field.

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Entamoeba histolytica causes invasive amoebiasis, an important neglected tropical disease with a significant global health impact. The pathogenicity and survival of E. histolytica and its reptilian equivalent, Entamoeba invadens, relies on its ability to exhibit efficient motility, evade host immune responses, and exploit host resources, all of which are governed by the actin cytoskeleton remodeling. Our study demonstrates the early origin and the regulatory role of TALE homeobox protein EiHbox1 in actin-related cellular processes. Several genes involved in different biological pathways, including actin dynamics are differentially expressed in EiHbox1 silenced cells. EiHbox1 silenced parasites showed disrupted F-actin organization and loss of cellular polarity. EiHbox1's presence in the anterior region of migrating cells further suggests its involvement in maintaining cellular polarity. Loss of polarized morphology of EiHbox1 silenced parasites leads to altered motility from fast, directionally persistent, and highly chemotactic to slow, random, and less chemotactic, which subsequently leads to defective aggregation during encystation. EiHbox1 knockdown also resulted in a significant reduction in phagocytic capacity and poor capping response. These findings highlight the importance of EiHbox1 of E. invadens in governing cellular processes crucial for their survival, pathogenicity, and evasion of the host immune system.

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The use of gene-editing tools, such as zinc finger nucleases, TALEN, and CRISPR/Cas, allows for the modification of physiological, morphological, and other characteristics in a wide range of crops to mitigate the negative effects of stress caused by anthropogenic climate change or biotic stresses. Importantly, these tools have the potential to improve crop resilience and increase yields in response to challenging environmental conditions. This review provides an overview of gene-editing techniques used in plants, focusing on the cultivated tomatoes. Several dozen genes that have been successfully edited with the CRISPR/Cas system were selected for inclusion to illustrate the possibilities of this technology in improving fruit yield and quality, tolerance to pathogens, or responses to drought and soil salinity, among other factors. Examples are also given of how the domestication of wild species can be accelerated using CRISPR/Cas to generate new crops that are better adapted to the new climatic situation or suited to use in indoor agriculture.

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The first TALE homeodomain transcription factor gene to be described in plants was maize knotted1 (kn1). Dominant mutations in kn1 disrupt leaf development, with abnormal knots of tissue forming in the leaf blade. kn1 was found to be expressed in the shoot meristem but not in a peripheral region that gives rise to leaves. Furthermore, KN1 and closely related proteins were excluded from initiating and developing leaves. These findings were a prelude to a large body of work wherein TALE homeodomain proteins have been identified as vital regulators of meristem homeostasis and organ development in plants. KN1 homologues are widely represented across land plant taxa. Thus, studying the regulation and mechanistic action of this gene class has allowed investigations into the evolution of diverse plant morphologies. This review will focus on the function of TALE homeodomain transcription factors in leaf development in eudicots. Here, we discuss how TALE homeodomain proteins contribute to a spectrum of leaf forms, from the simple leaves of Arabidopsis thaliana to the compound leaves of Cardamine hirsuta and species beyond the Brassicaceae.

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Touch sensation is primarily encoded by mechanoreceptors, called low-threshold mechanoreceptors (LTMRs), with their cell bodies in the dorsal root ganglia. Because of their great diversity in terms of molecular signature, terminal endings morphology, and electrophysiological properties, mirroring the complexity of tactile experience, LTMRs are a model of choice to study the molecular cues differentially controlling neuronal diversification. While the transcriptional codes that define different LTMR subtypes have been extensively studied, the molecular players that participate in their late maturation and in particular in the striking diversity of their end-organ morphological specialization are largely unknown. Here we identified the TALE homeodomain transcription factor Meis2 as a key regulator of LTMRs target-field innervation in mice. Meis2 is specifically expressed in cutaneous LTMRs, and its expression depends on target-derived signals. While LTMRs lacking Meis2 survived and are normally specified, their end-organ innervations, electrophysiological properties, and transcriptome are differentially and markedly affected, resulting in impaired sensory-evoked behavioral responses. These data establish Meis2 as a major transcriptional regulator controlling the orderly formation of sensory neurons innervating peripheral end organs required for light touch.

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KEY MESSAGE: TALE-based editors provide an alternative way to engineer the organellar genomes in plants. We update and discuss the most recent developments of TALE-based organellar genome editing in plants. Gene editing tools have been widely used to modify the nuclear genomes of plants for various basic research and biotechnological applications. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 editing platform is the most commonly used technique because of its ease of use, fast speed, and low cost; however, it encounters difficulty when being delivered to plant organelles for gene editing. In contrast, protein-based editing technologies, such as transcription activator-like effector (TALE)-based tools, could be easily delivered, expressed, and targeted to organelles in plants via Agrobacteria-mediated nuclear transformation. Therefore, TALE-based editors provide an alternative way to engineer the organellar genomes in plants since the conventional chloroplast transformation method encounters technical challenges and is limited to certain species, and the direct transformation of mitochondria in higher plants is not yet possible. In this review, we update and discuss the most recent developments of TALE-based organellar genome editing in plants.

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