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Adenosine deaminases acting on RNA (ADARs) are enzymes that catalyze the hydrolytic deamination of adenosine to inosine. The editing feature of ADARs has garnered much attention as a therapeutic tool to repurpose ADARs to correct disease-causing mutations at the mRNA level in a technique called site-directed RNA editing (SDRE). Administering a short guide RNA oligonucleotide that hybridizes to a mutant sequence forms the requisite dsRNA substrate, directing ADARs to edit the desired adenosine. However, much is still unknown about ADARs' selectivity and sequence-specific effects on editing. Atomic-resolution structures can help provide additional insight to ADARs' selectivity and lead to novel guide RNA designs. Indeed, recent structures of ADAR domains have expanded our understanding on RNA binding and the base-flipping catalytic mechanism. These efforts have enabled the rational design of improved ADAR guide strands and advanced the therapeutic potential of the SDRE approach. While no full-length structure of any ADAR is known, this review presents an exposition of the structural basis for function of the different ADAR domains, focusing on human ADAR2. Key insights are extrapolated to human ADAR1, which is of substantial interest because of its widespread expression in most human tissues.

Adenosine Deaminases Acting on RNA (ADARs) are enzymes that catalyze the conversion of adenosine to inosine in RNA duplexes. These enzymes can be harnessed to correct disease-causing G-to-A mutations in the transcriptome because inosine is translated as guanosine. Guide RNAs (gRNAs) can be used to direct the ADAR reaction to specific sites. Chemical modification of ADAR guide strands is required to facilitate delivery, increase metabolic stability, and increase the efficiency and selectivity of the editing reaction. Here, we show the ADAR reaction is highly sensitive to ribose modifications (e.g. 4'-C-methylation and Locked Nucleic Acid (LNA) substitution) at specific positions within the guide strand. Our studies were enabled by the synthesis of RNA containing a new, ribose-modified nucleoside analog (4'-C-methyladenosine). Importantly, the ADAR reaction is potently inhibited by LNA or 4'-C-methylation at different positions in the ADAR guide. While LNA at guide strand positions -1 and -2 block the ADAR reaction, 4'-C-methylation only inhibits at the -2 position. These effects are rationalized using high-resolution structures of ADAR-RNA complexes. This work sheds additional light on the mechanism of ADAR deamination and aids in the design of highly selective ADAR guide strands for therapeutic editing using chemically modified RNA.

There is great concern in equine sport over the potential use of pharmaceutical agents capable of editing the genome or modifying the expression of gene products. Synthetic oligonucleotides are short, single-stranded polynucleotides that represent a class of agents capable of modifying gene expression products with a high potential for abuse in horseracing. As these substances are not covered by most routine anti-doping analytical approaches, they represent an entire class of compounds that are not readily detectable. The nucleotide sequence for each oligonucleotide is highly specific, which makes targeted analysis for these agents problematic. Accordingly, we have developed a non-targeted approach to detect the presence of specific product ions that are not naturally present in ribonucleic acids. Briefly, serum samples were extracted using solid-phase extraction with a mixed-mode cartridge following the disruption of protein interactions to isolate the oligonucleotides. Following the elution and concentration steps, chromatographic separation was achieved utilizing reversed-phase liquid chromatography. Following an introduction to a Thermo Q Exactive HF mass spectrometer using electrospray ionization, analytes were detected utilizing a combination of full-scan, parallel reaction monitoring and all ion fragmentation scan modes. The limits of detection were determined along with the accuracy, precision, stability, recovery, and matrix effects using a representative 13mer oligonucleotide. Following method optimization using the 13mer oligonucleotide, the method was applied to successfully detect the presence of specific product ions in three unique oligonucleotide sequences targeting equine-specific transcripts.

Inosine (I), resulting from the deamination of adenosine (A), is a prominent modification in the human transcriptome. The enzymes responsible for the conversion of adenosine to inosine in human mRNAs are the ADARs (adenosine deaminases acting on RNA). Inosine modification introduces a layer of complexity to mRNA processing and function, as it can impact various aspects of RNA biology, including mRNA stability, splicing, translation, and protein binding. The relevance of this process is emphasized in the growing number of human disorders associated with dysregulated A-to-I editing pathways. Here, we describe the impact of the A-to-I conversion on the structure and stability of duplex RNA and on the consequences of this modification at different locations in mRNAs. Furthermore, we highlight specific open questions regarding the interplay between inosine formation in duplex RNA and the innate immune response.

In this study, we investigate how pre-service teachers' group dialogues emerged and intersected across time and space as students collaboratively constructed a video-based mind map to prepare for oral exams in a pedagogy course. The study was conducted as part of a design-based research project investigating the ways that video-based mind maps can support learning as both a collaborative activity and a classroom resource. We applied interaction analysis methods to recordings taken during the production of the mind map as well as the videos made by students within the mind map itself to analyze synchronous and asynchronous dialogues among group members as they viewed, recorded, and uploaded videos. The findings offer an in-depth understanding of how collaboration occurs in different space-time configurations within and across groups as mediated by video resources. We discuss how these findings contribute to computer-supported collaborative learning (CSCL) research on the ways collaboration can emerge across different levels of activity as well as the pedagogical implications for introducing video-based dialogues into the classroom.

The innate immune system relies on molecular sensors to detect distinctive molecular patterns, including viral double-stranded RNA (dsRNA), which triggers responses resulting in apoptosis and immune infiltration. Adenosine Deaminases Acting on RNA (ADARs) catalyze the deamination of adenosine (A) to inosine (I), serving as a mechanism to distinguish self from non-self RNA and prevent aberrant immune activation. Loss-of-function mutations in the ADAR1 gene are one cause of Aicardi Goutières Syndrome (AGS), a severe autoimmune disorder in children. Although seven out of the eight AGS-associated mutations in ADAR1 occur within the catalytic domain of the ADAR1 protein, their specific effects on the catalysis of adenosine deamination remain poorly understood. In this study, we carried out a biochemical investigation of four AGS-causing mutations (G1007R, R892H, K999N, and Y1112F) in ADAR1 p110 and truncated variants. These studies included adenosine deamination rate measurements with two different RNA substrates derived from human transcripts known to be edited by ADAR1 p110 (glioma-associated oncogene homologue 1 (hGli1), 5-hydroxytryptamine receptor 2C (5-HT2cR)). Our results indicate that AGS-associated mutations at two amino acid positions directly involved in stabilizing the base-flipped conformation of the ADAR-RNA complex (G1007R and R892H) had the most detrimental impact on catalysis. The K999N mutation, positioned near the RNA binding interface, altered catalysis contextually. Finally, the Y1112F mutation had small effects in each of the assays described here. These findings shed light on the differential effects of disease-associated mutations on adenosine deamination by ADAR1, thereby advancing our structural and functional understanding of ADAR1-mediated RNA editing.

Adenosine Deaminases Acting on RNA (ADARs) are members of a family of RNA editing enzymes that catalyze the conversion of adenosine into inosine in double-stranded RNA (dsRNA). ADARs' selective activity on dsRNA presents the ability to correct mutations at the transcriptome level using guiding oligonucleotides. However, this approach is limited by ADARs' preference for specific sequence contexts to achieve efficient editing. Substrates with a guanosine adjacent to the target adenosine in the 5' direction (5'-GA) are edited less efficiently compared to substrates with any other canonical nucleotides at this position. Previous studies showed that a G/purine mismatch at this position results in more efficient editing than a canonical G/C pair. Herein, we investigate a series of modified oligonucleotides containing purine or size-expanded nucleoside analogs on guide strands opposite the 5'-G (-1 position). The results demonstrate that modified adenosine and inosine analogs enhance editing at 5'-GA sites. Additionally, the inclusion of a size-expanded cytidine analog at this position improves editing over a control guide bearing cytidine. High-resolution crystal structures of ADAR:/RNA substrate complexes reveal the manner by which both inosine and size-expanded cytidine are capable of activating editing at 5'-GA sites. Further modification of these altered guide sequences for metabolic stability in human cells demonstrates that the incorporation of specific purine analogs at the -1 position significantly improves editing at 5'-GA sites.

Objetivo: Analisar a confi abilidade e a adesão à terapêutica medicamentosa em idosos. Os idosos, em sua maioria, apresentam doenças crônicas como a hipertensão arterial e, por isso, utilizam muitos medicamentos. A maioria é aposentada e se benefi cia dos programas de saúde e dos medicamentos ofertados pelo Ministério da Saúde (MS). Métodos: Os dados foram obtidos por meio de uma entrevista e da escolha visual entre três diferentes embalagens do medicamento diurético hidroclorotiazida 25 mg. Os dados foram estudados por meio da análise de conteúdo de Bardin. Resultados: Verifi cou-se que o medicamento fabricado pela indústria do governo (MS) foi o de primeira escolha, demonstrando grande credibilidade. Os argumentos foram: efi cácia, costume e gratuidade. O medicamento referência foi o de segunda escolha, devido ao custo fi nanceiro, e o medicamento similar foi a última escolha, por ser de marca desconhecida. Conclusão: Os resultados pressupõem que a indústria governamental precisa dar conta da demanda e manter o abastecimento nas unidades de saúde.

Purpose – This paper aims to present the vertical integration state-of-the-art and propose an expansion of the operations and supply chain management (OSCM) field by identifying gaps and bottlenecks. Design/methodology/approach – This paper uses a systematic literature review based on a sample of 173 OSCM field articles, collected from Scopus and Web of Science databases. Findings – There are no single factors, such as future costs, structures or skills development, in the decision to vertically integrate operations. It is necessary to combine the vision of production costs with the perspective of governance and transaction costs. In addition, it is essential to consider the competency perspective and its impact on capability building. Research limitations/implications – Few studies have attempted to understand how vertical integration is used in terms of OSCM research themes and theories. Vertical integration can help companies face challenges and serve as a potential solution for achieving better prices, demand control and quality management. Practical implications – The significant role of vertical integration mechanisms in supply chains is crucial for managers evaluating a firm's reconfiguration with more vertical operations. Policymakers interested in supporting the smoothness of vertical integration decisions in regulatory agencies play a key role as contingencies. Social implications – In times of global challenges, vertical integration is a strategy known to be more effective for firms to obtain a competitive advantage, making them more resilient. Originality/value – This paper addresses gaps in the vertical integration theme and provides insights for future research development.

Abstract Background Children in foster care who are newly placed with licensed or kinship caregivers are often vulnerable to increased behavior problems associated with trauma and social disruptions. When those issues are not addressed, children are susceptible to placement disruptions that exacerbate behavior problems. Few preventive interventions are available for foster and kinship caregivers, and none are designed to be delivered at the time of a child’s placement into the home. This study aims to examine the impact of the Chicago Parent Program adapted and customized for foster and kinship caregivers (CPP-FC), locally branded as Caregivers on Point, on caregiver stress, parenting confidence and strategies, children’s behaviors, and placement stability. Methods Caregivers (N = 300) are being recruited from a specialized foster care clinic that sees children and caregivers within five business days of a new placement. Upon completing baseline surveys and behavioral observation, caregiver-child dyads are randomized to receive CPP-FC (n = 150) or usual care (n = 150). Those in the treatment condition will complete 11 weekly sessions addressing child behavior management and caregiving approaches. A booster session will occur one month after the weekly sessions conclude. A mid-point assessment and behavioral observation will be collected four months after the baseline assessment for all participants, coinciding with the completion of the CPP-FC programming. At 6 months post-baseline, an end-of-study assessment will be collected. Administrative data will be extracted from the child welfare record to determine placement stability for the 12 months following enrolment. The primary outcome of interest is child behavior, indicated by changes in caregiver reports and objective ratings of behavior from observations, where raters are blinded to the treatment arm and timing of data collection. Secondary outcomes include placement stability and changes in caregiver stress and confidence in managing children’s behavior. Discussion If found to be effective, CPP-FC would be helpful for families involved with child welfare. It could be delivered by child welfare agencies, licensing and kinship navigator agencies, and foster care clinics and may be eligible for government reimbursement as a preventive intervention for children in foster care. Trial registration This study was prospectively registered with ClinicalTrials.gov, NCT06170047 .

KRASG12C inhibitors, such as sotorasib and adagrasib, have revolutionized cancer treatment for patients with KRASG12C-mutant tumors. However, patients receiving these agents as monotherapy often develop drug resistance. To address this issue, we evaluated the combination of the PAK4 inhibitor KPT9274 and KRASG12C inhibitors in preclinical models of pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer (NSCLC). PAK4 is a hub molecule that links several major signaling pathways and is known for its tumorigenic role in mutant Ras-driven cancers. We found that cancer cells resistant to KRASG12C inhibitor were sensitive to KPT9274-induced growth inhibition. Furthermore, KPT9274 synergized with sotorasib and adagrasib to inhibit the growth of KRASG12C-mutant cancer cells and reduce their clonogenic potential. Mechanistically, this combination suppressed cell growth signaling and downregulated cell-cycle markers. In a PDAC cell line-derived xenograft (CDX) model, the combination of a suboptimal dose of KPT9274 with sotorasib significantly reduced the tumor burden (P= 0.002). Similarly, potent antitumor efficacy was observed in an NSCLC CDX model, in which KPT9274, given as maintenance therapy, prevented tumor relapse following the discontinuation of sotorasib treatment (P= 0.0001). Moreover, the combination of KPT9274 and sotorasib enhances survival. In conclusion, this is the first study to demonstrate that KRASG12C inhibitors can synergize with the PAK4 inhibitor KPT9274 and combining KRASG12C inhibitors with KPT9274 can lead to remarkably enhanced antitumor activity and survival benefits, providing a novel combination therapy for patients with cancer who do not respond or develop resistance to KRASG12C inhibitor treatment.

Adenosine deaminases acting on RNA (ADARs) catalyze the hydrolytic deamination of adenosine to inosine in duplex RNA. The inosine product preferentially base pairs with cytidine resulting in an effective A-to-G edit in RNA. ADAR editing can result in a recoding event alongside other alterations to RNA function. A consequence of ADARs' selective activity on duplex RNA is that guide RNAs (gRNAs) can be designed to target an adenosine of interest and promote a desired recoding event. One of ADAR's main limitations is its preference to edit adenosines with specific 5' and 3' nearest neighbor nucleotides (e.g., 5' U, 3' G). Current rational design approaches are well-suited for this ideal sequence context, but limited when applied to difficult-to-edit sites. Here we describe a strategy for the in vitro evaluation of very large libraries of ADAR substrates (En Masse Evaluation of RNA Guides, EMERGe). EMERGe allows for a comprehensive screening of ADAR substrate RNAs that complements current design approaches. We used this approach to identify sequence motifs for gRNAs that enable editing in otherwise difficult-to-edit target sites. A guide RNA bearing one of these sequence motifs enabled the cellular repair of a premature termination codon arising from mutation of the MECP2 gene associated with Rett Syndrome. EMERGe provides an advancement in screening that not only allows for novel gRNA design, but also furthers our understanding of ADARs' specific RNA-protein interactions.

HER2-Low is defined as low levels of HER2 expression, based on a score of 1+ on immunohistochemical (IHC) assay or as an IHC score of 2+ and negative results on in situ hybridization (ISH or FISH). They are a heterogeneous population of breast cancers that vary in prognosis and sensitivity to systemic treatments. The frequency and clinical characteristics of pathogenic germline variants (PGVs) in HER2-Low breast cancer (BC) patients is not defined. We analyzed results from patients with BC who underwent multi-gene panel testing (MGPT) (maximum 145 genes) between 2018–2019. We reclassified HER-2 status accordingly. Relationships between the variables of interest were assessed by adopting the proportional regression Cox models. Of a total of 167 BC patients who underwent MGPT, half were hormone-receptor-positive. The median age was 45 years. About two thirds of the patients were in the earlier stage of BC. A total of 57% of the cases were reclassified as HER-2-negative or -Low. PGVs were found in 19% of the patients overall, as follows: seven BRCA1, four BRCA2, two ATM, one ATR, two CFTR, three CHEK2, one FANCA, one MERTK, one MLH1, three MUTYH, one RAD50, three RAD51C, one RECQL4, and two TP53 mutations. In HER2-Low, 26.5% of the patients had PGVs, and in the overall cohort, this was 19.8%. In conclusion, differences in the prevalence of deleterious germline mutations in HER2-Low BC patients compared to non-HER2-Low BC patients were identified. Similar alterations in BRCA were observed in this group of patients compared to the overall cohort. Germline genetic tests should be evaluated in larger cohorts of patients with HER2-Low status to better address the findings.

Abstract Fire plays a key role in grasslands, determining the distribution and evolution of species and boundaries with neighboring ecosystems. Evidence of community-wide responses to fire is largely based on taxonomic and functional descriptors, while the phylogenetic dimension is overlooked. Here we evaluated how the taxonomic and phylogenetic structure of grassland plant communities responded to a time since fire (TSF) gradient. We sampled 12 communities in Southern Brazil under varying TSF and calculated taxonomic species richness (S) and dominance (D), phylogenetic diversity (PD), and mean phylogenetic distances (MPD). We used Structural Equation Models to test the relationships between the environmental gradient and community descriptors. Communities with longer TSF presented higher PD and MPD but lower species richness and increased taxonomic dominance. These sites were dominated by monocots, specifically C4 grasses, but also presented exclusive clades, whereas recently-burned sites presented lower taxonomic dominance and more species distributed in a wider variety of clades. Our results indicate that these scenarios are interchangeable and dependent on fire management. Fire adaptation was not constrained by phylogenetic relatedness, contrasting with previous findings for tropical savannahs and indicating that temperate and tropical non-forest ecosystems from South America respond differently to fire, possibly due to different evolutionary histories.

Purpose – This teaching case aims to encourage reflection on the formulation of the strategic repositioning of an automotive dealership. Design/methodology/approach – Built on the teaching case method, it portrays the situation of the Premium Motors automotive dealership in the face of a strategic repositioning. Management and the board discuss ways to minimize the impact of the coronavirus disease 2019 (COVID-19) pandemic on sales figures. The situation presents the possibility of entering a new product segment. Findings – How to make consumers correctly understand the new positioning? The case is structured to enable reflection and teaching of marketing strategies, with each student having the possibility of putting themselves in the role of company managers. The environment and trajectory are also portrayed, broadening the perception of the studied company and providing devices for solving the emerging problems of the case. Originality/value – The case becomes a tool to promote knowledge, from the implementation to the management of strategic repositioning. Thus, the teaching notes offer directions on how professors can use the teaching case with their undergraduate and graduate students in disciplines related to strategy and marketing.

The term RNA editing refers to any structural change in an RNA molecule (e.g. insertion, deletion, or base modification) that changes its coding properties and is not a result of splicing. An important class of enzymes involved in RNA editing is the ADAR family (adenosine deaminases acting on RNA), which facilitate the deamination of adenosine (A) to inosine (I) in double-stranded RNA (dsRNA). Inosines are decoded as guanosines (G) in most cellular processes; hence, A-to-I editing can be considered an A-to-G substitution. Among the RNA editing enzymes, ADARs are of particular interest because a large portion of RNA editing events are due to A-to-I editing by the two catalytically active human ADARs (ADAR1 and ADAR2). ADARs have diverse roles in RNA processing, gene expression regulation, and innate immunity; and mutations in the ADAR genes and dysregulated ADAR activity have been associated with cancer, autoimmune diseases, and neurological disorders. A-to-I editing is also currently being explored for correcting disease-causing mutations in the RNA, where therapeutic guide oligonucleotides complementary to the target transcript are used to form a dsRNA substrate and site-specifically direct ADAR editing. Knowledge of the mechanism of ADAR-catalyzed reaction and the origin of its substrate selectivity will allow understanding of ADAR’s role in disease biology and expedite the process of developing ADAR-targeted therapeutics. Chemically modified oligonucleotides provide a versatile platform for modulating the activity and interrogating the structure, function, and selectivity of nucleic acid binding or modifying proteins. In this account, we provide an overview of oligonucleotide modifications that have allowed us to gain deeper understanding of ADAR’s molecular mechanisms, which we utilize in the rational design and optimization of ADAR activity modulators. First, we describe the use of the nucleoside analog 8-azanebularine (8-azaN) to generate high-affinity ADAR-RNA complexes for biochemical and biophysical studies with ADARs, with particular emphasis on X-ray crystallography. We then discuss key observations derived from the crystal structures of ADAR bound to 8-azaN-modified RNA duplexes and describe how these findings provided insight into ADAR editing optimization by introducing nucleoside modifications at various positions in synthetic guide strands. We also present the informed design of 8-azaN-modified RNA duplexes that selectively bind and inhibit ADAR1 but not the closely-related ADAR2 enzyme. Finally, we conclude with some open questions on ADAR structure and substrate recognition and share our current endeavors in the development of ADAR guide oligonucleotides and inhibitors.