Simultaneous Detection of Adenosine-to-Inosine Editing and N6-Methyladenosine at Identical RNA Sites through Deamination-Assisted Reverse Transcription Stalling.

Adenosine-to-inosine (A-to-I) editing and N6-methyladenosine (m6A) modifications are pivotal RNA modifications with widespread functional significance in physiological and pathological processes. Although significant effort has been dedicated to developing methodologies for identifying and quantifying these modifications, traditional approaches have often focused on each modification independently, neglecting the potential co-occurrence of A-to-I editing and m6A modifications at the same adenosine residues. This limitation has constrained our understanding of the intricate regulatory mechanisms governing RNA function and the interplay between different types of RNA modifications. To address this gap, we introduced an innovative technique called deamination-assisted reverse transcription stalling (DARTS), specifically designed for the simultaneous quantification of A-to-I editing and m6A at the same RNA sites. DARTS leverages the selective deamination activity of the engineered TadA-TadA8e protein, which converts adenosine residues to inosine, in combination with the unique property of Bst 2.0 DNA polymerase, which stalls when encountering inosine during reverse transcription. This approach enables the accurate quantification of A-to-I editing, m6A, and unmodified adenosine at identical RNA sites. The DARTS method is remarkable for its ability to directly quantify two distinct types of RNA modifications simultaneously, a capability that has remained largely unexplored in the field of RNA biology. By facilitating a comprehensive analysis of the co-occurrence and interaction between A-to-I editing and m6A modifications, DARTS opens new avenues for exploring the complex regulatory networks modulated by different RNA modifications.

Design, synthesis, and cell-based in vitro assay of deoxyinosine-mixed SATE-dCDN prodrugs that activate all common STING variants.

Developing therapeutic strategies to modulate the activity of all prevalent variants (wild-type, HAQ, R232H, AQ, and R293Q) of the stimulator of interferon genes (STING) is still of great interest to treating immune-related diseases. Herein, we synthesized six novel deoxyinosine-mixed deoxyribose cyclic dinucleotide prodrugs (SATE-dCDN) including a combination of hypoxanthine and other bases (A, U, C, T, and G) for a cell-based in vitro assay. The HPLC assay indicated that deoxyinosine-mixed SATE (S-acylthioalkyl ester)-dCDN prodrugs retained high serum stability. The IRF3-responsive luciferase assay in THP1-Lucia cells showed that the activity of the prodrugs with purine bases (SATE-3',3'-c-di-dIMP, SATE-3',3'-c-di-dIdAMP, and SATE-3',3'-c-di-dIdGMP) was higher than that of the prodrugs with pyrimidine bases (SATE-3',3'-c-di-dIdUMP, SATE-3',3'-c-di-dIdTMP, and SATE-3',3'-c-di-dIdCMP), among which prodrug 14a (SATE-3',3'-c-di-dIdAMP) with hypoxanthine and adenine bases exhibited the highest activity with an EC50 value of 0.046 µM. The IRF3 responsive dual-luciferase reporter assay in HEK293T cells transfected with plasmids expressing different STING variants further showed that prodrug 14a could activate all five most common hSTING variants, including the refractory hSTINGR232H and hSTINGQ variants. Furthermore, prodrug 14a also induced the production of the highest levels of mRNA of IFN-ß, CXCL10, IL-6 and TNF-α through STING-dependent IRF and NF-κB signaling pathways in THP-1 cells. These results suggested that the combination of deoxyinosine with a SATE-dCDN prodrug could modulate the broad-spectrum activity of all common STING variants.

Assistência fisioterapêutica na atenção primária à saúde infantil: uma revisão das experiências / Physical therapy in pediatric primary care: a review of experiences

OBJETIVO: Revisar as experiências de atenção fisioterapêutica dirigidas à população pediátrica descritas na literatura e analisar a produção de conhecimento sobre fisioterapia no contexto da atenção primária à saúde infantil (APSI). MÉTODOS: Foi realizada uma revisão sistemática conforme PRISMA, com busca nas seguintes bases de dados: MEDLINE, LILACS, SciELO, PubMed, Scopus, Cochrane; banco de teses da CAPES; e System for Information on Grey Literature in Europe (SIGLE). Foram utilizados os descritores "atenção primária à saúde", "fisioterapia", "lactente ou criança" e seus correspondentes na língua inglesa e espanhola, sem restrição de ano de publicação. RESULTADOS: Analisamos 13 artigos de seis países, reunidos em três eixos temáticos: dilemas profissionais (três artigos), competências e habilidades específicas para a APSI (sete artigos) e relatos de prática (quatro artigos). Os dilemas profissionais mencionados foram a ampliação do papel do fisioterapeuta para incluir ambientes comunitários, compartilhando a tomada de decisão com as famílias, e o trabalho em colaboração com outros serviços de saúde para identificar as necessidades da criança. As competências e habilidades citadas foram a identificação de sintomas clínicos e socioculturais para além das condições musculoesqueléticas, o diagnóstico fisioterapêutico precoce, a prevenção contra o uso excessivo de medicamentos e a capacidade de trabalhar em equipe. Os relatos de prática discorreram sobre estimulação em crianças com quadros neurológicos, tratamento respiratório e grupos com mães de crianças com esses acometimentos. CONCLUSÕES: O baixo número de estudos sugere desconhecimento quanto ao modo como a fisioterapia se insere na APSI e, provavelmente, quanto às habilidades profissionais necessárias nesse ambiente. Assim, são necessários mais estudos para fornecer dados sobre a área e um esforço de qualificação continuada por parte dos fisioterapeutas.

OBJECTIVE: To review pediatric physical therapy experiences described in the literature and to analyze the production of knowledge on physical therapy in the context of pediatric primary health care (PPHC). METHODS: A systematic review was conducted according to the PRISMA criteria. The following databases were searched: MEDLINE, LILACS, SciELO, PubMed, Scopus and Cochrane; Brazilian Ministry of Health's CAPES doctoral dissertations database; and System for Information on Grey Literature in Europe (SIGLE). The following search terms were used: ["primary health care" and ("physical therapy" or "physiotherapy") and ("child" or "infant")] and equivalent terms in Portuguese and Spanish, with no restriction on publication year. RESULTS: Thirteen articles from six countries were analyzed and grouped into three main themes: professional dilemmas (three articles), specific competencies and skills required in a PPHC setting (seven articles), and practice reports (four articles). Professional dilemmas involved expanding the role of physical therapists to encompass community environments and sharing the decision-making process with the family, as well as collaborative work with other health services to identify the needs of children. The competencies and skills mentioned in the literature related to the identification of clinical and sociocultural symptoms that go beyond musculoskeletal conditions, the establishment of early physical therapy diagnoses, prevention of overmedication, and the ability to work as team players. Practice reports addressed stimulation in children with neurological diseases, respiratory treatment, and establishing groups with mothers of children with these conditions. CONCLUSIONS: The small number of studies identified in this review suggests that there is little knowledge regarding the roles of physical therapists in PPHC and possibly regarding the professional abilities required in this setting. Therefore, further studies are required to provide data on the field, along with a continuing education effort on the part of physical therapists.