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Impaired control of the G1/S checkpoint allows initiation of DNA replication under non-permissive conditions. Unscheduled S-phase entry is associated with DNA replication stress, demanding for other checkpoints or cellular pathways to maintain proliferation. Here, we uncovered a requirement for ADARp150 to sustain proliferation of G1/S-checkpoint-defective cells under growth-restricting conditions. Besides its well-established mRNA editing function in inversely oriented short interspersed nuclear elements (SINEs), we found ADARp150 to exert a critical function in mitosis. ADARp150 depletion resulted in tetraploidization, impeding cell proliferation in mitogen-deprived conditions. Mechanistically we show that ADAR1 depletion induced aberrant expression of Cyclin B3, which was causative for mitotic failure and whole-genome duplication. Finally, we find that also in vivo ADAR1-depletion-provoked tetraploidization hampers tumor outgrowth.

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.

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.

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.

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 to inosine (A-to-I) RNA editing by ADAR1 has been implicated in maintaining self-tolerance, preventing autoimmunity, and mediating antiviral immunity. Foreign viral double-stranded RNA triggers rapid interferon response and activates ADAR1 in the host immune system. Emerging data points to a role of ADAR1 A-to-I editing in the inflammatory response associated with severe COVID-19 disease. We identify A-to-I editing events within human whole transcriptome data from SARS-CoV-2 infected individuals, non-infected individuals, and individuals with other viral illnesses from nasopharyngeal swabs. High levels of RNA editing in host cells are associated with low SARS-CoV-2 viral load (p = 9.27 E-06), suggesting an inhibitory effect of ADAR1 on viral infection. Additionally, we find differentially expressed genes associated with RNA-modifications and interferon response. Single cell RNA-sequencing analysis of SARS-CoV-2 infected nasopharyngeal swabs reveals that cytotoxic CD8 T cells upregulate ADAR1 in COVID-19 positive samples (p = 0.0269). We further reveal ADAR1 expression increases with CD4 and CD8 T cell activation, and knockdown of ADAR1 leads to apoptosis and aberrant IL-2 secretion. Together, our data suggests A-to-I RNA editing is required to maintain healthy homeostasis of activated T cells to combat SARS-CoV-2 infection.

BackgroundRNA-seq has brought forth significant discoveries regarding aberrations in RNA processing, implicating these RNA variants in a variety of diseases. Aberrant splicing and single nucleotide variants (SNVs) in RNA have been demonstrated to alter transcript stability, localization, and function. In particular, the upregulation of ADAR, an enzyme that mediates adenosine-to-inosine editing, has been previously linked to an increase in the invasiveness of lung adenocarcinoma cells and associated with splicing regulation. Despite the functional importance of studying splicing and SNVs, the use of short-read RNA-seq has limited the community's ability to interrogate both forms of RNA variation simultaneously.ResultsWe employ long-read sequencing technology to obtain full-length transcript sequences, elucidating cis-effects of variants on splicing changes at a single molecule level. We develop a computational workflow that augments FLAIR, a tool that calls isoform models expressed in long-read data, to integrate RNA variant calls with the associated isoforms that bear them. We generate nanopore data with high sequence accuracy from H1975 lung adenocarcinoma cells with and without knockdown of ADAR. We apply our workflow to identify key inosine isoform associations to help clarify the prominence of ADAR in tumorigenesis.ConclusionsUltimately, we find that a long-read approach provides valuable insight toward characterizing the relationship between RNA variants and splicing patterns.

BackgroundCase reportConclusionsDeficiency of adenosine deaminase 2 (DADA2) is a rare autosomal recessive autoinflammatory disorder associated with systemic vasculitis and bone marrow failure. Reported ophthalmic findings in DADA2 include optic neuritis, retinal artery occlusion, uveitis, and optic atrophy. We report the case of a child found to have bilateral cataracts.A three-year-old recent immigrant from Mexico with a diagnosis of DADA2 and transfusion-dependent anemia was referred to ophthalmology to screen for deferasirox-associated retinopathy in the setting of hemochromatosis. He was incidentally found to have bilateral posterior subcapsular cataracts with no other ophthalmic abnormalities. The child’s lab findings were significant for chronic hyperferritinemia, and his history was significant for over a year of oral prednisone use in Mexico.This is the first reported case of cataracts in a child with DADA2. While DADA2 is an autoinflammatory disorder, this child’s lack of uveitis suggests a non-inflammatory etiology. Hyperferritinemia is a known cause of cataracts and is common in DADA2, but the child’s history of oral steroid use in Mexico could also explain his cataracts. As pediatric cataracts have not otherwise been reported in DADA2, ophthalmologists should be aware of this possibility, especially in children with hyperferritinemia or a history of steroid use. [ABSTRACT FROM AUTHOR]

Obesity stands out as one of the most significant health problems in the modern world. The prevalence of high‐calorie diets (HCDs) globally exacerbates this condition. Throughout history, plants and plant‐derived food products have been utilized for medicinal purposes, demonstrating their efficacy in the treatment and prevention of various diseases. Gundelia tournefortii (GT), a plant of interest, is known to possess beneficial properties. Hence, this study aimed to investigate the immunotoxic and neurotoxic effects of two different doses of GT plant extract on the liver, brain, and heart tissues of obese rats. For this purpose, Wistar male rats were divided into four groups: “CG,” “HCDG,” “HCDGUN1,” and “HCDGUN2” At the conclusion of the study, adenosine deaminase (ADA) and myeloperoxidase (MPO) activities, as well as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) biomarkers, were evaluated in the liver, heart, and brain tissues. The study results revealed a statistically significant increase in ADA and MPO activities in the HCDG group compared to the CG group, alongside a significant decrease in the HCDGUN groups compared to the HCDG group. Regarding AChE and BChE activities, a statistically significant decrease was observed in the HCDG group compared to the CG group, whereas an increase was noted in the HCDGUN groups relative to the HCDG group, with the latter approaching values similar to those of the control group. In conclusion, the intake of GT plant extract exhibited positive effects on the immunotoxic and neurotoxic effects induced by HCD in rats with an experimental obesity model, as evidenced by tissue biomarker evaluations. [ABSTRACT FROM AUTHOR]

Summary: Dual base editors (DBEs) enable simultaneous A‐to‐G and C‐to‐T conversions, expanding mutation types. However, low editing efficiency and narrow targeting range limit the widespread use of DBEs in plants. The single‐strand DNA binding domain of RAD51 DBD can be fused to base editors to improve their editing efficiency. However, it remains unclear how the DBD affects dual base editing performance in plants. In this study, we generated a series of novel plant DBE‐SpGn tools consisting of nine constructs using the high‐activity cytidine deaminase evoFERNY, adenosine deaminase TadA8e and DBD in various fusion modes with the PAM‐flexible Streptococcus pyogenes Cas9 (SpCas9) nickase variant SpGn (with NG‐PAM). By analysing their editing performance on 48 targets in rice, we found that DBE‐SpGn constructs containing a single DBD and deaminases located at the N‐terminus of SpGn exhibited the highest editing efficiencies. Meanwhile, constructs with deaminases located at the C‐terminus and/or multiple DBDs failed to function normally and exhibited inhibited editing activity. We identified three particularly high‐efficiency dual base editors (C‐A‐SpGn, C‐A‐D‐SpGn and A‐C‐D‐SpGn), named PhieDBEs (Plant high‐efficiency dual base editors), capable of producing efficient dual base conversions within a narrow editing window (M5 ~ M9, M = A/C). The editing efficiency of C‐A‐D‐SpGn was as high as 95.2% at certain target sites, with frequencies of simultaneous C‐to‐T and A‐to‐G conversions as high as 81.0%. In summary, PhieDBEs (especially C‐A‐D‐SpGn) can produce diverse mutants and may prove useful in a wide variety of applications, including plant functional genomics, precise mutagenesis, directed evolution and crop genetic improvement, among others. [ABSTRACT FROM AUTHOR]

Introduction: Dynamic cellular and molecular adaptations in early life significantly impact health and disease. Upon birth, newborns are immediately challenged by their environment, placing urgent demands on the infant immune system. Adenosine deaminases (ADAs) are enzymatic immune modulators present in two isoforms - ADA-1 and ADA-2. Infants exhibit low ADA activity, resulting in high plasma adenosine concentrations and a consequent antiinflammatory/anti-Th1 bias. While longitudinal studies of plasma ADA have been conducted in infants in The Gambia (GAM), little is known regarding ADA trajectories in other parts of the world. Methods: Herein, we characterized plasma ADA activity in an infant cohort in Papua New Guinea (PNG; n=83) and compared to ontogeny of ADA activity in a larger cohort in GAM (n=646). Heparinized peripheral blood samples were collected at day of life (DOL) 0, DOL7, DOL30, and DOL128. Plasma ADA-1, ADA-2, and total ADA activities were measured by chromogenic assay. Results: Compared to GAM infants, PNG infants had significantly lower ADA-1 (0.9-fold), ADA-2 (0.42-fold), and total ADA (0.84-fold) activities at birth which converged by DOL30. Discussion: Overall, discovery of a distinct baseline and a consistent pattern of increasing plasma ADA activity in early life in two genetically and geographically distinct populations validates and extends previous findings on the robustness of early life immune ontogeny. [ABSTRACT FROM AUTHOR]

RNA editing is a highly conserved process. Adenosine deaminase acting on RNA (ADAR) mediated deamination of adenosine (A-to-I editing) is associated with human disease and immune checkpoint control. Functional implications of A-to-I editing are currently of broad interest to academic and industrial research as underscored by the fast-growing number of clinical studies applying base editors as therapeutic tools. Analyzing the dynamics of A-to-I editing, in a biological or therapeutic context, requires the sensitive detection of differential A-to-I editing, a currently unmet need. We introduce the local differential editing index (LoDEI) to detect differential A-to-I editing in RNA-seq datasets using a sliding-window approach coupled with an empirical q value calculation that detects more A-to-I editing sites at the same false-discovery rate compared to existing methods. LoDEI is validated on known and novel datasets revealing that the oncogene MYCN increases and that a specific small non-coding RNA reduces A-to-I editing. Functional implications of A-to-I editing is of broad interest to scientific research. Here, authors introduce the local differential editing index (LoDEI) to detect differential A-to-I editing in RNAseq datasets using a sliding-window approach coupled with an empirical q value calculation. [ABSTRACT FROM AUTHOR]

Background: China is experiencing rapid growth in its population of older adults, which may lead to increased susceptibility to tuberculous pleural effusion (TPE) due to age-related changes in the immune system. This study aimed to investigate the diagnostic potential of multiple biomarkers in individuals aged 60 years and above with pleural effusion. Methods: A total of 519 adult patients from Ningbo First Hospital were included in the study, with 7 biomarkers and their ratios in serum and pleural effusion analyzed using logistic regression analysis. Effusion Adenosine Deaminase(ADA), γ-Interferon Release Assay(IGRA), and Effusion lactatedehy drogenase(LDH)/Effusion ADA were identified as valuable parameters for differentiating TPE from non-TPE, particularly in individuals aged 60 years and older. Results: Effusion ADA, IGRA, and Effusion LDH/Effusion ADA were identified as valuable parameters for the differential diagnosis of TPE from non-TPE, showing good diagnostic performance in individuals aged 60 years and older. The combined diagnosis of these three indexes achieved the highest diagnostic accuracy for TPE in this age group, with an AUC of 0.925, sensitivity of 85.23%, and specificity of 89.57%. Conclusions: Overall, the study highlights the importance of using multiple indicators for a combined diagnosis to improve diagnostic efficacy in detecting tuberculous pleurisy in older individuals as for young patients. [ABSTRACT FROM AUTHOR]

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The engineered TadA variants used in cytosine base editors (CBEs) present distinctive advantages, including a smaller size and fewer off-target effects compared to cytosine base editors that rely on natural deaminases. However, the current TadA variants demonstrate a preference for base editing in DNA with specific motif sequences and possess dual deaminase activity, acting on both cytosine and adenosine in adjacent positions, limiting their application scope. To address these issues, we employ TadA orthologs screening and multi sequence alignment (MSA)-guided protein engineering techniques to create a highly effective cytosine base editor (aTdCBE) without motif and adenosine deaminase activity limitations. Notably, the delivery of aTdCBE to a humanized mouse model of Duchenne muscular dystrophy (DMD) mice achieves robust exon 55 skipping and restoration of dystrophin expression. Our advancement in engineering TadA ortholog for cytosine editing enriches the base editing toolkits for gene-editing therapy and other potential applications. Engineered TadA variants in cytosine base editors (CBEs) offer advantages like smaller size and reduced off-target effects. Here, authors develop an advanced CBE (aTdCBE) through ortholog screening and protein engineering, successfully enabling exon skipping in Duchenne muscular dystrophy model. [ABSTRACT FROM AUTHOR]

Introduction: T2DM is associated with cell-mediated immune responses and abnormal T-lymphocyte function, further linked to insulin deficiency. Hence this study aims to determine the activity of serum total ADA and correlate these parameters with glycemic control, and biochemical variables in type 2 diabetic individuals. Aims and Objectives: Aim: To estimate serum adenosine deaminase and its correlation with glycemic status in diabetes mellitus. Objectives: To estimate the serum adenosine deaminase levels in type 2 diabetes mellitus patients. Inclusion Criteria: Individuals with diagnosed T2DM in the age group 35 to 70 years. Exclusion Criteria: Individuals with a history of tuberculosis, rheumatoid arthritis, viral hepatitis, and HIV and Patients on insulin therapy. Results: In this study, 41 cases were males and 59 were females. ADA activity was significantly higher in uncontrolled (Group C) diabetic patients (55.428±3.736U/L) compared to the controlled (Group B) population (42.63±4.59 U/L) and non-diabetics (22.0581±5.1968 U/L) with p value 0.001. Patients who have a longer duration of diabetes history had higher serum ADA levels than newly diagnosed patients with significant differences, p < 0.001. Conclusion: This study's findings clearly show that Adenosine deaminase (ADA) levels are elevated in type 2 diabetics, and the positive correlation of ADA with higher glycemic control suggests that ADA may serve as a prognostic factor in T2DM. ADA had a significant positive correlation with HbA1c, which is regarded as a good marker for long-term glycemic control. [ABSTRACT FROM AUTHOR]

Esophageal cancer is mainly divided into esophageal adenocarcinoma and esophageal squamous cell carcinoma (ESCC). China is one of the high-incidence areas of esophageal cancer, of which about 90% are ESCC. The deubiquitinase USP38 has been reported to play significant roles in several biological processes, including inflammatory responses, antiviral infection, cell proliferation, migration, invasion, DNA damage repair, and chemotherapy resistance. However, the role and mechanisms of USP38 in ESCC development remain still unclear. Furthermore, although many substrates of USP38 have been identified, few upstream regulatory factors of USP38 have been identified. In this study, we found that USP38 was significantly upregulated in esophageal cancer tissues. Knockdown of USP38 inhibited ESCC growth. USP38 stabilized itself through auto-deubiquitylation. In addition, we demonstrate that adenosine deaminase acting on RNA (ADAR) could enhance the stability of USP38 protein and facilitate USP38 auto-deubiquitylation by interacting with USP38 in an RNA editing-independent manner. ADAR inhibition of ESCC cell proliferation depended on USP38. In summary, these results highlight that the potential of targeting the ADAR-USP38 axis for ESCC treatment. [ABSTRACT FROM AUTHOR]

Simple Summary: The colostrum and milk of sows may contain substances that could be used to evaluate immunity and welfare in pigs. This study aimed to quantify in these fluids four analytes associated with immunity: total adenosine deaminase (tADA) and its isoenzymes, myeloperoxidase (Mpx), calprotectin (S100A8/A9) and calgranulin (S100A12) and two analytes which are linked to welfare: alpha-amylase and cortisol. In addition, it aimed to evaluate how these analytes change during lactation and how they relate to each other and to immunoglobulin G (IgG) and immunoglobulin A (IgA). All the assays tested were able to quantify the analytes in a precise and accurate way. In addition, the analytes showed different dynamics through lactation, with adenosine deaminase being more concentrated in colostrum than in milk, while myeloperoxidase was more active in mature milk than in colostrum. Alpha-amylase was correlated with both IgG and IgA and S100A8/A9 was correlated with S100A12 and Mpx. Overall, the immune system and welfare analytes of this study can be measured in sow colostrum and milk samples and could have a potential use as biomarkers in these fluids. Colostrum is a mammary secretion released from the time of farrowing to 36 h post-farrowing. After this time and during all the rest of lactation, the mammary secretion is considered milk. The objectives of this study were: (1) to perform an analytical validation in the colostrum and milk of sows of assays for four analytes related to immunity: total ADA (tADA) and its isoenzymes (ADA1 and ADA2), myeloperoxidase (Mpx), calprotectin, and calgranulin, and two analytes related to welfare: cortisol and alpha-amylase. (2) To evaluate the changes in these analytes during lactation (3) To assess the correlations between these new analytes, as well as with IgG and IgA. In the analytical validation, all the assays were precise and accurate. When changes during lactation were evaluated, the concentration of tADA and ADA2 was found to be higher in colostrum than in milk (p < 0.02), while the activity of Mpx was observed to be higher in mature milk than in colostrum (p < 0.03). Furthermore, cortisol and alpha-amylase activity were found to be higher in colostrum compared to mature milk (p < 0.04 and p < 0.0001, respectively). Regarding the relation between analytes, alpha-amylase showed a significant correlation with both IgG and IgA and calprotectin was correlated with calgranulin and Mpx. Further studies should be performed to elucidate the possible practical application of the analytes evaluated in this study as biomarkers of colostrum and milk in sows. [ABSTRACT FROM AUTHOR]

As one of the most important economic traits for domestic animal husbandry, skeletal muscle is regulated by an intricate molecular network. Adenosine deaminase acting on RNA (ADAR1) involves various physiological processes and diseases, such as innate immunity and the development of lung adenocarcinoma, breast cancer, gastric cancer, etc. However, its role in skeletal muscle growth requires further clarification. Here, we explored the functions of ADAR1 in the myogenic process of goat skeletal muscle satellite cells (MuSCs). The ADAR1 transcripts were noticeably enriched in goat visceral tissues compared to skeletal muscle. Additionally, its levels in slow oxidative muscles like the psoas major and minor muscles were higher than in the fast oxidative glycolytic and fast glycolytic muscles. Among the two common isoforms from ADAR1, p110 is more abundant than p150. Moreover, overexpressing ADAR1 enhanced the proliferation and myogenic differentiation of MuSCs. The mRNA-seq performed on MuSCs' knockdown of ADAR1 obtained 146 differentially expressed genes (DEGs), 87 upregulated and 59 downregulated. These DEGs were concentrated in muscle development and process pathways, such as the MAPK and cAMP signaling pathways. Furthermore, many DEGs as the key nodes defined by protein–protein interaction networks (PPI), including STAT3, MYH3/8, TGFβ2, and ACTN4, were closely related to the myogenic process. Finally, RNA immunoprecipitation combined with qPCR (RIP-qPCR) showed that ADAR1 binds to PAX7 and MyoD mRNA. This study indicates that ADAR1 promotes the myogenic development of goat MuSCs, which provides a useful scientific reference for further exploring the ADAR1-related regulatory networks underlying mammal skeletal muscle growth. [ABSTRACT FROM AUTHOR]

Neuropathic pain (NPP) is a devastating and unbearable painful condition. As prevailing treatment strategies have failed to mitigate its complications, there remains a demand for effective therapies. Electroacupuncture (EA) has proved a potent remedial strategy in NPP management in humans and mammals. However, past studies have investigated the underlying mechanism of the analgesic effects of EA on NPP, focusing primarily on adenosine receptors in peripheral tissues. Herein, we elucidate the role of the adenosine (Adora-3) signaling pathway in mediating pain relief through EA in the central nervous system, which is obscure in the literature and needs exploration. Specific pathogen-free (SPF) male adult mice (C57BL/6 J) were utilized to investigate the effect of EA on adenosine metabolism (CD73, ADA) and its receptor activation (Adora-3), as potential mechanisms to mitigate NPP in the central nervous system. NPP was induced via spared nerve injury (SNI). EA treatment was administered seven times post-SNI surgery, and lumber (L4–L6) spinal cord was collected to determine the molecular expression of mRNA and protein levels. In the spinal cord of mice, following EA application, the expression results revealed that EA upregulated (p < 0.05) Adora-3 and CD73 by inhibiting ADA expression. In addition, EA triggered the release of adenosine (ADO), which modulated the nociceptive responses and enhanced neuronal activation. Meanwhile, the interplay between ADO levels and EA-induced antinociception, using an Adora-3 agonist and antagonist, showed that the Adora-3 agonist IB-MECA significantly increased (p < 0.05) nociceptive thresholds and expression levels. In contrast, the antagonist MRS1523 exacerbated neuropathic pain. Furthermore, an upregulated effect of EA on Adora-3 expression was inferred when the Adora-3 antagonist was administered, and the EA treatment increased the fluorescent intensity of Adora-3 in the spinal cord. Taken together, EA effectively modulates NPP by regulating the Adora-3 signaling pathway under induced pain conditions. These findings enhance our understanding of NPP management and offer potential avenues for innovative therapeutic interventions. [ABSTRACT FROM AUTHOR]

Background: The simultaneous involvement of the pleura and peritoneum with tuberculosis in the absence of pulmonary foci is an uncommon condition that may lead physicians to misdiagnose. Case Presentation: Herein, we present a Persian male adult who manifested with epigastric pain, weakness, and a history of pleuritic chest pain two months prior to admission. The findings of the physical examination included vital signs within the normal range, unilateral fine crackle in the lung, abdominal distension with positive shifting dullness, and fluid wave test. Analysis of the ascitic fluid revealed a Serumascites albumin gradient (SAAG) of less than 1.1g/dl, indicating a non-portal condition. The results of the acid-fast bacilli (AFB) staining as well as the TB polymerase chain reaction (PCR) test were negative. However, the adenosine deaminase (ADA) level was 44 IU/L. A chest CT scan revealed mediastinal lymph node enlargement and pleural thickening with loculated pleural effusion. Three acid-fast bacilli smear of morning sputum were sent, and all three were negative. An abdominopelvic CT scan showed multiple periaortic and mesenteric lymph nodes of varying sizes with mesenteric haziness and accumulation of effusion in the peritoneal cavity. Eventually, peritoneal biopsy, the gold standard, was performed, which revealed multiple granulomatous lesions and areas of caseous necrosis surrounded by Langerhans giant cells and epithelioid cells. Conclusion: It is worth noting that in cases of ascites and pleural thickening, especially in patients with poor socioeconomic status, simultaneous pleural and peritoneal TB should be considered, especially in third-world countries. [ABSTRACT FROM AUTHOR]

Objective: Rare disease. Background: Constrictive pericarditis occurs due to chronic pericardial inflammation and adherence of the cardiac pericardial layer. Etiologies include toxins, infection, cardiac surgery, and idiopathic causes. Outside the United States, the most common cause of constrictive pericarditis is tuberculosis (TB). Constrictive pericarditis is the most severe complication of tuberculous pericardial disease. Case Report: A 31-year-old man who recently immigrated to the United States presented with a 2-week history of constitutional symptoms, dyspnea, and pleuritic chest pain. Physical examination was pertinent for bilateral lower extremity pitting edema, decreased bilateral breath sounds, and jugular venous distension. Transthoracic echocardiogram revealed a left ventricular ejection fraction of 45%, pericardial thickening, and an exaggerated septal bounce. Right heart catheterization showed discordant and concordant right ventricular pressure tracings. Cardiac magnetic resonance imaging revealed bilateral pleural effusions and circumferential pericardial thickening. Thoracocentesis was significant for an exudative effusion, with elevated adenosine deaminase levels. Subsequent QuantiFERON-TB Gold testing was positive, and he underwent elective pericardiectomy. Pericardial histopathology revealed necrotizing caseating granulomas. He was discharged on a 6-month course of rifampicin, isoniazid, pyrazinamide, and ethambutol therapy, with close multidisciplinary care team outpatient follow-up. Conclusions: This case highlights the importance of a high index of clinical suspicion for tuberculous pericarditis in patients presenting with constitutional and heart failure symptoms and a relevant travel history, to ensure prompt diagnosis and treatment. This case also reflects the importance of coordination of care between cardiology, infectious disease, pathology, and cardiothoracic surgery teams in the management of tuberculous constrictive pericarditis. [ABSTRACT FROM AUTHOR]

The progression of kidney disease varies among individuals, but a general methodology to quantify disease timelines is lacking. Particularly challenging is the task of determining the potential for recovery from acute kidney injury following various insults. Here, we report that quantitation of post-transcriptional adenosine-to-inosine (A-to-I) RNA editing offers a distinct genome-wide signature, enabling the delineation of disease trajectories in the kidney. A well-defined murine model of endotoxemia permitted the identification of the origin and extent of A-to-I editing, along with temporally discrete signatures of double-stranded RNA stress and adenosine deaminase isoform switching. We found that A-to-I editing of antizyme inhibitor 1 (AZIN1), a positive regulator of polyamine biosynthesis, serves as a particularly useful temporal landmark during endotoxemia. Our data indicate that AZIN1 A-to-I editing, triggered by preceding inflammation, primes the kidney and activates endogenous recovery mechanisms. By comparing genetically modified human cell lines and mice locked in either A-to-I-edited or uneditable states, we uncovered that AZIN1 A-to-I editing not only enhances polyamine biosynthesis but also engages glycolysis and nicotinamide biosynthesis to drive the recovery phenotype. Our findings implicate that quantifying AZIN1 A-to-I editing could potentially identify individuals who have transitioned to an endogenous recovery phase. This phase would reflect their past inflammation and indicate their potential for future recovery [ABSTRACT FROM AUTHOR]

Background: The clinical significance of the presence or absence of Mycoplasma pneumoniae (MP) in pleural effusion in Mycoplasma pneumoniae pneumonia (MPP) children has not yet been elucidated. Herein, we investigated the clinical implication of pleural fluid MP positive in children with MPP. Methods: A total of 165 MPP children with pleural effusion requiring thoracocentesis were enrolled in this study. They were subsequently divided into two groups according to the presence or absence of MP in pleural effusion, namely positive group (n = 38) and negative group (n = 127). Information on their clinical manifestations, laboratory findings, radiological characteristics and treatment modalities was retrospectively collected from medical chart reviews. Results: The length of hospitalization (15.00 (10.75–19.25) vs. 11.00 (9.00–14.00) days, p=0.001) and total course of illness (23.00 (18.00–28.00) vs. 20.00 (17.00–24.00) days, p=0.010) were significantly longer in the positive group than in the negative group. The occurrence of pericardial effusion (23.7% vs. 7.9%, p=0.017), atelectasis (73.7% vs. 53.5%, p=0.027) and necrotizing pneumonia (23.7% vs. 7.9%, p=0.017) were more frequent in the positive group compared to the negative group. The levels of neutrophil percentages (82.35% (75.40%-85.78%) vs. 72.70% (64.30%-79.90%), p<0.001), C-reactive protein (CRP) (71.12 (37.75–139.41) vs. 31.15 (13.54–65.00) mg/L, p<0.001), procalcitonin (PCT) (0.65 (0.30–3.05) vs. 0.33 (0.17–1.13) ng/ml, p=0.005), serum lactate dehydrogenase (LDH) (799.00 (589.00–1081.50) vs. 673.00 (503.00–869.00) U/L, p=0.009), D-dimer (6.21 (3.37–16.11) vs. 3.32 (2.12–6.62) mg/L, p=0.001) on admission were significantly higher in the positive group than in the negative group. These pronounced differences significantly contributed to the identification of MPP with MP positive pleural effusion, as evidenced by the ROC curve analysis. Marked elevations in adenosine deaminase (49.25 (36.20–60.18) vs. 36.20 (28.10–46.50) U/L, p<0.001) and LDH levels (2298.50 (1259.75–3287.00) vs. 1199.00 (707.00–1761.00) U/L, p<0.001) were observed in pleural fluid of the positive group when compared to the negative group. Meanwhile, the number of patients on low molecular weight heparin (LMWH) therapy (9 (23.7%) vs. 12 (9.4%), p=0.028) was higher in the positive group. Multivariate logistic regression analysis revealed that D-dimer > 7.33 mg/L was significantly associated with the incidence of MP positive pleural effusion in MPP (OR=3.517). Conclusions: The presence of MP in pleural fluid in MPP children with pleural effusion indicated a more serious clinical course. D-dimer > 7.33 mg/L was a related factor for MP positive pleural effusion in MPP. The results of the present study would help in the creation of a therapeutic plan and prediction of the clinical course of MPP in children. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB) is a serious global public health problem. Detecting the changes of adenosine deaminase (ADA), interleukin (IL)-1ß, IL-2, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) released from macrophages and monocytes is greatly valuable in the diagnosis of TB. Hence, this study was designed to determine the optimal cut-off points of ADA, IL-1ß, IL-2, TNF-α, and IFN-γ, allowing to differentiate pleural TB (PLTB), peritoneal TB (PTB), and meningeal TB (MTB). The study included 386 patients with pleural effusion, 47 patients with peritoneal effusion, and 134 patients with encephalitis and meningoencephalitis suspect of TB. ADA was determined by an enzyme kinetics method; cytokine concentration was measured by a test based on the competitive enzyme-linked immunosorbent assay principle. Sensitivity, specificity, and positive and negative predictive value were calculated and described. In the aspiration fluid, ADA activity was greatly different between PLTB, PTB, and MTB groups with values of 37.5 U/L, 30.5 U/L, and 8.1 U/L and sensitivity and specificity of 87.7% and 83.4%; 100% and 88.9%; 97.3% and 98.6%, respectively. Similar, IFN-γ concentrations were distinctively different between PLTB, PTB, and MTB patients (with values of 120 pg/mL, 200 pg/mL, and 30 pg/mL with sensitivity and specificity of 80.3% and 80.9%; 80.0% and 92.6%; 98.6% and 61.7%, respectively). The determination of ADA and IFN-γ levels in the aspiration fluid is potentially considered a highly sensitive and specific test for differentiating PLTB, PTB, and MTB. [ABSTRACT FROM AUTHOR]

Objective • This study aimed to analyze the diagnostic efficacy of serum biomarkers in liver cirrhosis patients categorized by Child-Pugh scores. Methods • An observational cross-sectional study design was employed. A total of 110 liver cirrhosis patients, classified according to Child-Pugh scores and 60 healthy individuals were included in this study. Serum levels of adenosine deaminase (ADA), adiponectin (APN), matrix metalloproteinase-2 (MMP-2), alkaline phosphatase (ALP), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were measured. Results • The levels of ADA, APN, MMP-2, ALP, ALT, and AST were significantly higher in the study group compared to the control group (P < .05). Furthermore, these levels increased with the severity of liver cirrhosis, with higher levels observed in patients with Child-Pugh class C. The positive diagnostic rates for joint detection in Child-Pugh class A, B, and C were 93.75% (30/32), 100% (34/34), and 100% (44/44), respectively. Conclusions • Combined detection of serum biomarkers improves the diagnostic efficacy of liver cirrhosis. The diagnostic rates were higher when considering Child-Pugh scores, with the highest rates observed in class C. [ABSTRACT FROM AUTHOR]

Hydrocephalus, a major complication in tuberculous meningitis (TBM) patients, often necessitates treatment via ventriculoperitoneal shunt (VPS). However, post-VPS, some patients develop a complication called contralateral isolated lateral ventricle (CILV), leading to persistent hydrocephalus symptoms. This study aims to evaluate cerebrospinal fluid (CSF) parameters in predicting CILV occurrence post-VPS in adult TBM patients. A retrospective analysis was conducted, focusing on the relationship between preoperative CSF parameters and the development of CILV in 40 adult TBM patients who underwent VPS. The study compared CSF parameters from lumbar puncture after admission with those from ventricular CSF post-external ventricular drainage tube insertion. CILV was observed in 6 of the 40 patients following VPS. Statistical analysis showed no significant difference between the CSF parameters obtained via lumbar and ventricular punctures. Notably, the mean CSF glucose level in patients with CILV was significantly lower (1.92 mmol/L) compared to the non-CILV group (3.03 mmol/L). Conversely, the median adenosine deaminase (ADA) level in the CILV group was higher (5.69 U/L) compared to the non-CILV group (3.18 U/L). The optimal cutoff values for CSF glucose and ADA levels were 1.90 mmol/L and 4.80 U/L, respectively, with a sensitivity of 66.67% and 83.33% and a specificity of 88.24% and 79.41%. The study identified elevated ADA levels and decreased glucose levels in CSF as potential risk factors for CILV development in adult TBM patients post-VPS. These findings suggest the necessity for more tailored surgical approaches, in patients with altered CSF parameters to mitigate the risk of CILV. [ABSTRACT FROM AUTHOR]

Severe combined immunodeficiency secondary to adenosine deaminase deficiency is rare. The deficiency of this enzyme results in the accumulation of substrates in the tissues, including the brain. Clinical signs of neurological involvement may include seizures, neurodevelopmental disorders, hypotonia, and sensorineural hearing loss. Hematopoietic stem cell transplantation corrects the failure of the immune system but not the neurological involvement. To describe the spectrum of neurological complications identified in a series of children with severe combined immunodeficiency due to adenosine deaminase deficiency. Additionally, we propose a neurological approach including electrophysiological, radiological, and neurocognitive studies to address this group of children in an efficient and timely manner. A descriptive, observational, retro-, and prospective analysis of patients with a confirmed immunological diagnosis seen between 1996 and 2021 and referred to the Department of Neurology for neurological evaluation was conducted. Ten patients met the inclusion criteria. The median age at diagnosis was 4 months (range, 1-36 months). All patients had neurodevelopmental delay with hypotonia in six, language delay in three, sensorineural hearing loss in four, and spastic paraparesis in one patient. Two children developed an epileptic syndrome, consisting of generalized epilepsy in one and focal epilepsy in the other. Neuroimaging showed brain calcifications in the basal ganglia and/or centrum semiovale in four patients and enlarged subarachnoid spaces in two other patients. In this pediatric series, the rate of neurological involvement associated with abnormalities on neuroimaging was high. Although this involvement could be related to accumulation of adenosine metabolites in the central nervous system, the possibility of associated chronic infections should be ruled out. Given the neurological manifestations, it is important to involve the pediatric neurologist in the multidisciplinary follow-up team. [ABSTRACT FROM AUTHOR]

Simple Summary: Metritis, a condition affecting up to 20% of cows after giving birth, negatively impacts animal welfare and dairy farm profitability by reducing productivity and reproduction. This study compared the biochemical profiles of healthy cows and those with metritis, analyzing 25 salivary and 31 serum analytes alongside various health parameters. Key findings showed that cows with metritis had elevated levels of certain biomarkers related to stress, inflammation, and metabolism in both saliva and serum. Specifically, eight salivary biomarkers including adenosine deaminase (ADA) and haptoglobin (Hp) and eight serum biomarkers including ADA, Hp, and serum amyloid A (SAA) were significantly higher in affected cows. In contrast, six biomarkers including total esterase (TEA) and albumin were lower in the serum. This study highlights the potential of using saliva as a non-invasive source for identifying biomarkers in cows with metritis, offering a new approach to diagnose and manage this condition. Metritis affects 5–20% of cows after parturition, negatively impacting animal welfare and the profitability of dairy farms, increasing culling rates and costs, and decreasing productivity and reproduction rates. This study compared the results of a comprehensive biochemical panel consisting of 25 salivary and 31 serum analytes between healthy cows (n = 16) and cows with metritis (n = 12). Descriptive parameters such as depression, rectal temperature, body condition score (BCS), heart rate, respiratory rate, mucous color, ruminal motility, vaginal discharge, milk production, and complete hematology analyses were also assessed for comparative purposes. The biochemistry analytes comprised five analytes related to stress, five to inflammation, five to oxidative status, and nineteen to general metabolism. The two-way ANOVA analysis revealed that, in saliva, eight biomarkers (lipase, adenosine deaminase (ADA), haptoglobin (Hp), total proteins, g-glutamyl transferase (gGT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and creatine kinase (CK)) were significant higher in cows with metritis. In serum, eight biomarkers (ADA, Hp, serum amyloid A (SAA), fibrinogen, ferritin, AOPPs/albumin ratio, non-esterified fatty acids (NEFAs), and bilirubin) were significantly higher in cows with metritis, whereas six (total esterase (TEA), albumin, urea, lactate, phosphorus, and calcium) were lower. Of the total number of 23 biomarkers that were measured in both saliva and serum, significant positive correlations between the two biofluids were found for six of them (Hp, FRAP, CUPRAC, AOPPs, urea, and phosphorus). Urea showed an R = 0.7, and the correlations of the other analytes were weak (R < 0.4). In conclusion, cows with metritis exhibited differences in biomarkers of stress, inflammation, cellular immune system, and general metabolism in both salivary and serum biochemistry profiles. These changes were of different magnitudes in the two biofluids. In addition, with the exception of ADA and Hp, the analytes that showed changes in the saliva and serum profiles of cows affected by metritis were different. Overall, this report opens a new window for the use of saliva as potential source of biomarkers in cows with metritis. [ABSTRACT FROM AUTHOR]

Diabetes Type 2 is the most common type of diabetes, a common disorder of glucose homeostasis and accounts for 90% of cases. The prevalence of diabetes type 2 is increasing. Adenosine deaminase is an enzymatic polymorphism that plays an important role in modulating the biological activity of insulin. It seems that excessive activity of the adenosine A1 receptor has caused adiposity in diabetes type 2. In this study, we examined the correlation of ADA enzyme with diabetes type 2. This investigation was performed on 80 men and women between 40 and 80 years old in District 2 of Tehran with diabetes. Venous blood samples were collected after 12 hours of fasting blood was centrifuged. Then fasting blood glucose and HbA1c, Triglyceride, and total Cholesterol were measured for enzyme activity respectively by COBAS MIRA. Insulin was measured by ELISA and serum ADA enzyme activity was measured by photometry. The results of this study were done by SPSS software. A significant increase in serum ADA levels was observed in diabetic patients compared with the control group. A positive correlation was observed between ADA activity and FBS and HbA1c. The amount of HOMA-IR in diabetics was higher than in the control group, but no positive correlation was observed between serum levels of ADA and HOMA-IR. The enzyme adenosine deaminase can act as an immunological marker and the results of this study show that diabetes is associated with increased T cell activation markers and immune disequilibrium. Serum ADA level has a positive correlation with glycemic control status in patients.

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.

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.

Background: Tuberculosis (TB) often leads to pleural effusion, particularly prevalent in developing nations such as India. There has been a global rise in TB cases. Although lymphocytic predominant fluid is commonly associated with tubercular pleural effusion, it is essential to note that not all lymphocytic predominant fluids indicate TB. The diagnosis of pleural TB has benefited significantly from the use of biochemical markers. Conventional bacteriological methods are not very useful in diagnosing tubercular effusion and rarely identify Mycobacterium tuberculosis in pleural fluid. Owing to diagnostic difficulties, newer investigations, such as TB polymerase chain reaction (TB-PCR), adenosine deaminase (ADA) and culture, are amongst the most recent techniques currently used due to the challenges associated with diagnosis. Aims: This study aimed to measure the sensitivity and specificity of TB-PCR and compare them with those of ADA and TB cultures for suspected TB pleural effusion. Methods: This study included 50 patients diagnosed with pleural effusion who underwent pleural fluid analysis. Patients exhibiting exudative effusion with lymphocyte predominance also underwent a pleural biopsy. Pleural fluid ADA levels were also measured, and TB-PCR tests were conducted. Results: Eighteen patients were confirmed to have TB by biopsy. ADA was both sensitive and specific at 67% and 62.5%, respectively. However, PCR showed a sensitivity of 16.6% and a specificity of 100%. Conclusion: This study found a statistically significant association (P < 0.05) between ADA levels and distinguishing pleural effusion, which is tubercular in origin, from non-tubercular effusion. Therefore, the pleural ADA estimate appears to have the potential to be a reliable test for diagnosing TB pleural effusion. It has sufficient sensitivity and specificity while being cost-effective and easily executable compared to pleural biopsy. Our study also compared the sensitivity of PCR with pleural biopsy and discovered that PCR was more specific and less sensitive.

Epilepsy is a common chronic neurological disorder affecting all age groups. A significant portion of children with epilepsy develop drug-resistant seizures. These children are at risk of cognitive and behavioral comorbidities and death. Some clinical features provide important information about the prognosis of epilepsy. However, currently, there is no objective biochemical indicator associated with refractory epilepsy. This study aimed to determine whether serum alpha-synuclein (αS), pglycoprotein (P-gp), and adenosine deaminase activity (ADA) were biochemical risk factors for refractory epilepsy.

Base editors (BEs) are a recent generation of genome editing tools that couple a cytidine or adenosine deaminase activity to a catalytically impaired Cas9 moiety (nCas9) to enable specific base conversions at the targeted genomic loci. Given their strong application potential, BEs are under active developments toward greater levels of efficiency and safety. Here, a previously overlooked nCas9‐centric strategy is explored for enhancement of BE. Based on a cytosine BE (CBE), 20 point mutations associated with nCas9‐target interaction are tested. Subsequently, from the initial positive X‐to‐arginine hits, combinatorial modifications are applied to establish further enhanced CBE variants (1.1–1.3). Parallel nCas9 modifications in other versions of CBEs including A3A‐Y130F‐BE4max, YEE‐BE4max, CGBE, and split‐AncBE4max, as well as in the context of two adenine BEs (ABE), likewise enhance their respective activities. The same strategy also substantially improves the efficiencies of high‐fidelity nCas9/BEs. Further evidence confirms that the stabilization of nCas9‐substrate interactions underlies the enhanced BE activities. In support of their translational potential, the engineered CBE and ABE variants respectively enable 82% and 25% higher rates of editing than the controls in primary human T‐cells. This study thus demonstrates a highly adaptable strategy for enhancing BE, and for optimizing other forms of Cas9‐derived tools. [ABSTRACT FROM AUTHOR]

The mechanisms of adenosine and specific adenosine receptor subtypes in promoting penile rehabilitation remain unclear. Single‐cell RNA sequencing of human corpus cavernosum, adenosine deaminase (ADA) and adenosine receptors knock‐out mice (ADA−/−, A1−/−, A2a−/−, A2b−/−, and A3−/−), and primary corpus cavernosum smooth muscle cells are used to determine receptor subtypes responsible for adenosine‐induced erection. Three rat models are established to characterize refractory erectile dysfunction (ED): age‐related ED, bilateral cavernous nerve crush related ED (BCNC), and diabetes mellitus‐induced ED. In single‐cell RNA sequencing data, the corpus cavernosum of ED patients show a decrease in adenosine A1, A2a and A2b receptors. In vivo, A2b receptor knock‐out abolishes adenosine‐induced erection but not that of A1, A2a, or A3 receptor. Under hypoxic conditions in vitro, activating the A2b receptor increases HIF‐1α and decreases PDE5 expression. In refractory ED models, activating the A2b receptor with Bay 60–6583 improves erectile function and down‐regulates HIF‐1α and TGF‐β. Administering Dipyridamole (40 mg Kg−1) to BCNC rats improve penile adenosine levels and erectile function. Our study reveals that the A2b receptor mediates adenosine‐induced penile erection. Activating the A2b receptor promotes penile rehabilitation of refractory ED by alleviating hypoxia and fibrosis. [ABSTRACT FROM AUTHOR]

Background. Adenosine deaminase deficiency 2 (DADA2) is an autoinflammatory disorder, caused by the CECR1 gene mutation. The major clinical manifestations include recurrent vasculitis, neurological disorders such as stroke, hematologic abnormalities, and immunodeficiency. As reported in previous studies, DADA2 may be manifested by ischemic or hemorrhagic strokes. This disorder also includes various hematological manifestations (pure red cell aplasia, pancytopenia, hemolytic anemia, and pancytopenia with bone marrow involvement). Case Presentation. In this case report, we present the clinical and immunological findings of two unrelated patients with DADA2. The first patient was a 7‐year‐old female who experienced recurrent neurological symptoms such as vertigo, tinnitus, hearing loss, and right‐sided hemiparesis. Her brain magnetic resonance imaging (MRI) revealed a left‐sided stroke, and she responded well to antitumor necrosis factor alpha agents and plasmapheresis. The second patient was a 6‐year‐old female who had recurrent fever and bicytopenia, aphthous lesions, cervical lymphadenopathy, and elevated liver enzymes. We also discussed the strategies used to manage the clinical manifestations in these two DADA2 patients. Conclusion. In this case report, we discussed two cases with DADA2 deficiency and their respective manifestations. The first case showed neurological symptoms while the second case had hematological symptoms. Although there is no established treatment for DADA2 due to its rarity, steroids are commonly used to treat this disorder. Antitumor necrosis factor is also effective in controlling the symptoms, especially the neurological ones. In cases where there is no appropriate response to these treatments, hematopoietic stem cell transplantation can be beneficial. [ABSTRACT FROM AUTHOR]

Glioblastoma (GB) is a lethal brain tumor that rapidly adapts to the dynamic changes of the tumor microenvironment (TME). Mesenchymal stem/stromal cells (MSCs) are one of the stromal components of the TME playing multiple roles in tumor progression. GB progression is prompted by the immunosuppressive microenvironment characterized by high concentrations of the nucleoside adenosine (ADO). ADO acts as a signaling molecule through adenosine receptors (ARs) but also as a genetic and metabolic regulator. Herein, the effects of high extracellular ADO concentrations were investigated in a human glioblastoma cellular model (U343MG) and MSCs. The modulation of the purinome machinery, i.e., the ADO production (CD39, CD73, and adenosine kinase [ADK]), transport (equilibrative nucleoside transporters 1 (ENT1) and 2 (ENT2)), and degradation (adenosine deaminase [ADA]) were investigated in both cell lines to evaluate if ADO could affect its cell management in a positive or negative feed‐back loop. Results evidenced a different behavior of GB and MSC cells upon exposure to high extracellular ADO levels: U343MG were less sensitive to the ADO concentration and only a slight increase in ADK and ENT1 was evidenced. Conversely, in MSCs, the high extracellular ADO levels reduced the ADK, ENT1, and ENT2 expression, which further sustained the increase of extracellular ADO. Of note, MSCs primed with the GB‐conditioned medium or co‐cultured with U343MG cells were not affected by the increase of extracellular ADO. These results evidenced how long exposure to ADO could produce different effects on cancer cells with respect to MSCs, revealing a negative feedback loop that can support the GB immunosuppressive microenvironment. These results improve the knowledge of the ADO role in the maintenance of TME, which should be considered in the development of therapeutic strategies targeting adenosine pathways as well as cell‐based strategies using MSCs. [ABSTRACT FROM AUTHOR]

Inosine is a nucleotide resulting from the deamination of adenosine in RNA. This chemical modification process, known as RNA editing, is typically mediated by a family of double-stranded RNA binding proteins named Adenosine Deaminase Acting on dsRNA (ADAR). While the presence of ADAR orthologs has been traced throughout the evolution of metazoans, the existence and extension of RNA editing have been characterized in a more limited number of animals so far. Undoubtedly, ADAR-mediated RNA editing plays a vital role in physiology, organismal development and disease, making the understanding of the evolutionary conservation of this phenomenon pivotal to a deep characterization of relevant biological processes. However, the lack of direct high-throughput methods to reveal RNA modifications at single nucleotide resolution limited an extended investigation of RNA editing. Nowadays, these methods have been developed, and appropriate bioinformatic pipelines are required to fully exploit this data, which can complement existing approaches to detect ADAR editing. Here, we review the current literature on the "bioinformatics for inosine" subject and we discuss future research avenues in the field. [ABSTRACT FROM AUTHOR]

Background: Venlafaxine is frequently prescribed for patients with depression. To control the concentration of venlafaxine within the therapeutic window for the best treatment effect, a model to predict venlafaxine concentration is necessary. Aim: Our objective was to develop a prediction model for venlafaxine concentration using real-world evidence based on machine learning and deep learning techniques. Method: Patients who underwent venlafaxine treatment between November 2019 and August 2022 were included in the study. Important variables affecting venlafaxine concentration were identified using a combination of univariate analysis, sequential forward selection, and machine learning techniques. Predictive performance of nine machine learning and deep learning algorithms were assessed, and the one with the optimal performance was selected for modeling. The final model was interpreted using SHapley Additive exPlanations. Results: A total of 330 eligible patients were included. Five influential variables that affect venlafaxine concentration were venlafaxine daily dose, sex, age, hyperlipidemia, and adenosine deaminase. The venlafaxine concentration prediction model was developed using the eXtreme Gradient Boosting algorithm (R2 = 0.65, mean absolute error = 77.92, root mean square error = 93.58). In the testing cohort, the accuracy of the predicted concentration within ± 30% of the actual concentration was 73.49%. In the subgroup analysis, the prediction accuracy was 69.39% within the recommended therapeutic range of venlafaxine concentration within ± 30% of the actual value. Conclusion: The XGBoost model for predicting blood concentration of venlafaxine using real-world evidence was developed, guiding the adjustment of regimen in clinical practice. [ABSTRACT FROM AUTHOR]

It is well established that sevoflurane exposure leads to widespread neuronal cell death in the developing brain. Adenosine deaminase acting on RNA-1 (ADAR1) dependent adenosine-to-inosine (A-to-I) RNA editing is dynamically regulated throughout brain development. The current investigation is designed to interrogate the contributed role of ADAR1 in developmental sevoflurane neurotoxicity. Herein, we provide evidence to show that developmental sevoflurane priming triggers neuronal pyroptosis, apoptosis and necroptosis (PANoptosis), and elicits the release of inflammatory factors including IL-1β, IL-18, TNF-α and IFN-γ. Additionally, ADAR1-P150, but not ADAR1-P110, depresses cellular PANoptosis and inflammatory response by competing with Z-DNA/RNA binding protein 1 (ZBP1) for binding to Z-RNA in the presence of sevoflurane. Further investigation demonstrates that ADAR1-dependent A-to-I RNA editing mitigates developmental sevoflurane-induced neuronal PANoptosis. To restore RNA editing, we utilize adeno-associated virus (AAV) to deliver engineered circular ADAR-recruiting guide RNAs (cadRNAs) into cells, which is capable of recruiting endogenous adenosine deaminases to promote cellular A-to-I RNA editing. As anticipated, AAV-cadRNAs diminishes sevoflurane-induced cellular Z-RNA production and PANoptosis, which could be abolished by ADAR1-P150 shRNA transfection. Moreover, AAV-cadRNAs delivery ameliorates developmental sevoflurane-induced spatial and emotional cognitive deficits without influence on locomotor activity. Taken together, these results illustrate that ADAR1-P150 exhibits a prominent role in preventing ZBP1-dependent PANoptosis through A-to-I RNA editing in developmental sevoflurane neurotoxicity. Application of engineered cadRNAs to rectify the compromised ADAR1-dependent A-to-I RNA editing provides an inspiring direction for possible clinical preventions and therapeutics. [ABSTRACT FROM AUTHOR]

Adenosine has garnered significant attention as a potential target for overcoming tumor immune evasion. It exacerbates the immunosuppressive tumor microenvironment (TME) by incapacitating various protective immune cells. However, inhibitors targeting adenosine receptors are limited by incomplete blockade effects and off‐target toxicity, necessitating the exploration of more effective strategies. Inspired by the native biologic function of adenosine deaminase (ADA) in hydrolyzing adenosine, a bioactive catalytic nanovesicle (ENV@FeS) is designed to reverse adenosine immunosuppressive function. The nanovesicle is constructed through the serial extrusion of erythrocytes abundant with ADA, coupled with the synthesis of FeS complex in situ using Fe2+ in erythrocytes via H2S gas introduction. Notably, the bioactive nanovesicles not only promote tumor targeting and induce immunogenic cell death by the FeS‐induced photothermal effect, but also hinder the adenosine pathway by metabolizing adenosine into immunopotentiator inosine, further boosting antitumor immunity. In vivo, ENV@FeS efficiently increases the infiltration of cytotoxic T lymphocytes and M1 macrophages while reducing the production of regulatory T cells and myeloid‐derived suppressor cells, resulting in significant inhibition of tumor growth. Together, the biomimetic nanovesicle‐mediated adenosine metabolic regulation, in synergy with photothermal therapy, leads to a marked improvement in the immunogenic TME and enhances the therapeutic efficacy of photoimmunotherapy. [ABSTRACT FROM AUTHOR]

• Eleven UGT SNP loci were simultaneously assessed and a comprehensive analysis of their effects conducted, along with clinical factors, on mycophenolic acid metabolism. • Patients who carry the UGT1A9 rs2741049 TT genotype might be at a greater risk of a lower dose-adjusted MPAG C 0. • UGT2B7 rs7662029 GG genotype carriers may face a higher risk of AcMPAG C 0 after high-dose adjustment. This study aimed to evaluate the effects of UDP-glucuronosyltransferase (UGT) polymorphisms on mycophenolic acid (MPA) metabolism in renal transplant patients. A total of 11 single nucleotide polymorphisms (SNPs) of UGT1A1, UGT1A7, UGT1A8, UGT1A9, UGT1A10 , and UGT2B7 were genotyped in 79 renal transplant patients. The associations of SNPs and clinical factors with dose-adjusted MPA area under the plasma concentration-time curve (AUC/D), the dose-adjusted plasma concentration (C 0 /D) of 7-O-MPA-glucuronide (MPAG), and the dose-adjusted plasma concentration (C 0 /D) of acyl MPAG (AcMPAG) were analyzed. In the univariate analysis, UGT1A1 rs4148323, age, and anion gap were associated with MPA AUC/D. MPA AUC/D was higher in patients with the GA genotype of UGT1A1 rs4148323 compared to patients with the GG genotype. UGT1A1 rs4148323, UGT1A9 rs2741049 and clinical factors, including age, serum total bilirubin, adenosine deaminase, anion gap, urea, and creatinine, were associated with MPAG C 0 /D. UGT2B7 rs7438135, UGT2B7 rs7439366, and UGT2B7 rs7662029 also were associated with AcMPAG C 0 /D. Multiple linear regression analysis showed that UGT1A9 rs2741049 and indirect bilirubin were negatively correlated with MPAG C 0 /D (P =.001; P =.039), and UGT2B7 rs7662029 was positively correlated with AcMPAG C 0 /D (P =.008). This study demonstrates a significant influence of UGT1A9 rs2741049 and UGT2B7 rs7662029 polymorphisms on the metabolism of MPA in vivo. [ABSTRACT FROM AUTHOR]

Although lack of ADAR (adenosine deaminase acting on RNA) orthologs, genome-wide A-to-I editing occurs specifically during sexual reproduction in a number of filamentous ascomycetes, including Fusarium graminearum and Neurospora crassa. Unlike ADAR-mediated editing in animals, fungal A-to-I editing has a strong preference for hairpin loops and U at −1 position, which leads to frequent editing of UAG and UAA stop codons. Majority of RNA editing events in fungi are in the coding region and cause amino acid changes. Some of these editing events have been experimentally characterized for providing heterozygote and adaptive advantages in F. graminearum. Recent studies showed that FgTad2 and FgTad3, 2 ADAT (adenosine deaminase acting on tRNA) enzymes that normally catalyze the editing of A34 in the anticodon of tRNA during vegetative growth mediate A-to-I mRNA editing during sexual reproduction. Stage specificity of RNA editing is conferred by stage-specific expression of short transcript isoforms of FgTAD2 and FgTAD3 as well as cofactors such as AME1 and FIP5 that facilitate the editing of mRNA in perithecia. Taken together, fungal A-to-I RNA editing during sexual reproduction is catalyzed by ADATs and it has the same sequence and structural preferences with editing of A34 in tRNA. [ABSTRACT FROM AUTHOR]

This article discusses deficiency of adenosine deaminase 2 (DADA2), a rare autoinflammatory disorder characterized by inflammation due to inappropriate activation of the innate immune system. The article emphasizes the importance of early recognition and confirmatory testing to avoid unnecessary invasive testing and treatments. It presents a case of DADA2 with an unusual presentation of skeletal muscle vasculitis. The article also discusses the broad phenotypic spectrum of DADA2 and the availability of targeted therapies for treatment. The authors highlight the significance of genetic testing and next-generation sequencing technology in accurate diagnosis and treatment of autoinflammatory disorders. [Extracted from the article]

The dynamic process of Drosophila spermatogenesis involves asymmetric division, mitosis, and meiosis, which ultimately results in the production of mature spermatozoa. Disorders of spermatogenesis can lead to infertility in males. ADAR (adenosine deaminase acting on RNA) mutations in Drosophila cause male infertility, yet the causative factors remain unclear. In this study, immunofluorescence staining was employed to visualize endogenous ADAR proteins and assess protein levels via fluorescence-intensity analysis. In addition, the early differentiation disorders and homeostatic alterations during early spermatogenesis in the testes were examined through quantification of transit-amplifying region length, counting the number of GSCs (germline stem cells), and fertility experiments. Our findings suggest that deletion of ADAR causes testicular tip transit-amplifying cells to accumulate and become infertile in older male Drosophila. By overexpressing ADAR in early germline cells, male infertility can be partially rescued. Transcriptome analysis showed that ADAR maintained early spermatogenesis homeostasis through the bone-morphogenetic-protein (BMP) signaling pathway. Taken together, these findings have the potential to help explore the role of ADAR in early spermatogenesis. [ABSTRACT FROM AUTHOR]

Ascites is the pathological accumulation of fluid within the peritoneal cavity. It often occurs as results of liver cirrhosis, malignant neoplasia, tuberculous infection, cardiac insufficiency, renal diseases, etc. Determining the etiology is an essential step in the management of patients with new-onset ascites. Abdominal paracentesis with appropriate ascitic fluid analysis is probably the most cost-effective method of determining the cause of ascites. We performed a literature search of PubMed and identified articles published in the field of ascites, to evaluate diagnostic values of various parameters in defining the etiologies of ascites and then provides diagnostic algorithm for patients with new-onset ascites. In patients with ascites, the constituent ratio of underlying etiology varies between developed and developing countries. It is a challenge to define the etiologies of ascites in developing countries. Routine ascitic fluid analysis should include the serum ascites albumin gradient (SAAG), total protein concentration, cell count and differential. Optional ascitic fluid analysis includes cholesterol, fluid culture, cytology, tumor markers, lactate dehydrogenase, adenosine deaminase (ADA), triglyceride, amylase, glucose, brain natriuretic peptide (BNP), etc. Our review evaluated diagnostic values of the above parameters in defining the etiologies of ascites. Diagnostic algorithm established in this review would provide a practical and convenient diagnostic strategy for clinicians in diagnosing patients with new-onset ascites. [ABSTRACT FROM AUTHOR]

Purpose: Patients with adenosine deaminase 1 deficient severe combined immunodeficiency (ADA-SCID) are initially treated with enzyme replacement therapy (ERT) with polyethylene glycol-modified (PEGylated) ADA while awaiting definitive treatment with hematopoietic stem cell transplant (HSCT) or gene therapy. Beginning in 1990, ERT was performed with PEGylated bovine intestinal ADA (ADAGEN®). In 2019, a PEGylated recombinant bovine ADA (Revcovi®) replaced ADAGEN following studies in older patients previously treated with ADAGEN for many years. There are limited longitudinal data on ERT-naïve newborns treated with Revcovi. Methods: We report our clinical experience with Revcovi as initial bridge therapy in three newly diagnosed infants with ADA-SCID, along with comprehensive biochemical and immunologic data. Results: Revcovi was initiated at twice weekly dosing (0.2 mg/kg intramuscularly), and monitored by following plasma ADA activity and the concentration of total deoxyadenosine nucleotides (dAXP) in erythrocytes. All patients rapidly achieved a biochemically effective level of plasma ADA activity, and red cell dAXP were eliminated within 2–3 months. Two patients reconstituted B-cells and NK-cells within the first month of ERT, followed by naive T-cells one month later. The third patient reconstituted all lymphocyte subsets within the first month of ERT. One patient experienced declining lymphocyte counts with improvement following Revcovi dose escalation. Two patients developed early, self-resolving thrombocytosis, but no thromboembolic events occurred. Conclusion: Revcovi was safe and effective as initial therapy to restore immune function in these newly diagnosed infants with ADA-SCID, however, time course and degree of reconstitution varied. Revcovi dose may need to be optimized based on immune reconstitution, clinical status, and biochemical data. [ABSTRACT FROM AUTHOR]

RNA therapeutics have had a tremendous impact on medicine, recently exemplified by the rapid development and deployment of mRNA vaccines to combat the COVID-19 pandemic. In addition, RNA-targeting drugs have been developed for diseases with significant unmet medical needs through selective mRNA knockdown or modulation of pre-mRNA splicing. Recently, RNA editing, particularly antisense RNA-guided adenosine deaminase acting on RNA (ADAR)-based programmable A-to-I editing, has emerged as a powerful tool to manipulate RNA to enable correction of disease-causing mutations and modulate gene expression and protein function. Beyond correcting pathogenic mutations, the technology is particularly well suited for therapeutic applications that require a transient pharmacodynamic effect, such as the treatment of acute pain, obesity, viral infection, and inflammation, where it would be undesirable to introduce permanent alterations to the genome. Furthermore, transient modulation of protein function, such as altering the active sites of enzymes or the interface of protein-protein interactions, opens the door to therapeutic avenues ranging from regenerative medicine to oncology. These emerging RNA-editing-based toolsets are poised to broadly impact biotechnology and therapeutic applications. Here, we review the emerging field of therapeutic RNA editing, highlight recent laboratory advancements, and discuss the key challenges on the path to clinical development.

IntroductionDynamic cellular and molecular adaptations in early life significantly impact health and disease. Upon birth, newborns are immediately challenged by their environment, placing urgent demands on the infant immune system. Adenosine deaminases (ADAs) are enzymatic immune modulators present in two isoforms – ADA-1 and ADA-2. Infants exhibit low ADA activity, resulting in high plasma adenosine concentrations and a consequent anti-inflammatory/anti-Th1 bias. While longitudinal studies of plasma ADA have been conducted in infants in The Gambia (GAM), little is known regarding ADA trajectories in other parts of the world.MethodsHerein, we characterized plasma ADA activity in an infant cohort in Papua New Guinea (PNG; n=83) and compared to ontogeny of ADA activity in a larger cohort in GAM (n=646). Heparinized peripheral blood samples were collected at day of life (DOL) 0, DOL7, DOL30, and DOL128. Plasma ADA-1, ADA-2, and total ADA activities were measured by chromogenic assay.ResultsCompared to GAM infants, PNG infants had significantly lower ADA-1 (0.9-fold), ADA-2 (0.42-fold), and total ADA (0.84-fold) activities at birth which converged by DOL30.DiscussionOverall, discovery of a distinct baseline and a consistent pattern of increasing plasma ADA activity in early life in two genetically and geographically distinct populations validates and extends previous findings on the robustness of early life immune ontogeny.