RESUMEN

Guanine deaminase (GD)plays important roles in the diagnosis of liver function. However, there is no totally rapid and simple for the eatimation of GD activity in clinical application. Herein, we have constructed an enzymatic assay system with highly sensitive and strong stability for quantification of GD activity by highly double enzyme-coupling (xanthine oxidase and uric acid oxidase) and adding compound stabilizer in GD kit. In this study, we validated parameters, including reagent blank, sensitivity, accuracy, inter-batch difference, intra-batch difference, linear range. Furthermore, composite stabilizers, containing gentamicin sulfate, bovine serum albumin, and mannitol, were selected to improve stability of GD kit during long-term storage. The experimental results showed that the absorbance of the reagent blank was <0.2, the mean recovery rate was 103 %, the inter-batch and intra-batch diffeerence were <15 %, The linearity range was 0 U/L-50 U/L (R2 > 0.99). All indicators met the kit requirements for clinical applications. When gentamicin sulfate, bovine serum albumin, and mannitol were used as a stabilizer, the kit remained stable for 12 months without significant loss of enzymatic activity. These results indicated that GD kit possesses high sensitivity and strong stability, which can be used for routine biochemical applications and is of great significance for the diagnosis and differential diagnosis of liver diseases.

RESUMEN

BACKGROUND: Cancer metastasis is the leading cause of cancer-related deaths, underscoring the importance of understanding its underlying mechanisms. Hepatocellular carcinoma (HCC), a highly malignant type of cancer, was selected as our research model. MATERIAL AND METHODS: We aimed to develop high-metastatic cell lines using in vitro and in vivo selection strategies and identify critical metastasis-related genes through microarray analyses by comparing them with parental cells. RESULTS: Our results showed that the high-metastatic cell lines exhibited significantly stronger invasion abilities than parental cells. Microarray analyses identified cytidine deaminase (CDA), a gene associated with systemic chemotherapy resistance, as one of the overexpressed genes in the high-metastatic cells. Data analysis from The Cancer Genome Atlas Program revealed that while CDA is downregulated in HCC, patients with high CDA expression tend to have poorer prognoses. Cell models confirmed that CDA overexpression enhances cell migration and invasion, whereas CDA knockdown inhibits these abilities. Investigating the key molecules involved in the epithelial-mesenchymal transition (EMT), we found that CDA overexpression increases the expression of fascin, N-cadherin, ß-catenin, and snail while decreasing E-cadherin expression. Conversely, CDA knockdown produced opposite results. Additionally, we discovered that CDA regulates NF-κB signaling, which controls the expression of N-cadherin, thereby promoting the invasion capability of HCC cells. CONCLUSIONS: We isolated highly metastatic cells and identified potential HCC metastasis-related genes. CDA promotes cell invasion by regulating EMT through the NF-κB pathway. Future studies are warranted to explore the potential of CDA as a biomarker for prognosis and therapeutic decision-making.

RESUMEN

BACKGROUND: Double-hit lymphoma (DHL) with c-MYC gene translocation is highly aggressive and has a poor prognosis. In DHL cells, activation-induced cytidine deaminase (AID) promotes antibody class switch recombination (CSR), ultimately leading to c-MYC gene translocation caused by Myc/IgH DNA double-strand breaks. However, currently there is still no method to suppress the expression of AID. METHODS: In this study, we compared the clinical significance of AID expression in DHL, Additionally, two human double-hit lymphoma cell lines were used to analyze the effect of imatinib mesylate on c-MYC in vitro, and the therapeutic effect was also evaluated in xenograft mouse models. RESULTS: Imatinib mesylate downregulated the AID and c-MYC proteins in patients with chronic myelogenous leukemia associated with DHL. In addition, imatinib mesylate reduced AID and c-MYC expression in SU-DHL-4 and OCI-Ly18 DHL cells. Imatinib mesylate exerted significant inhibitory effects on the proliferation and metastasis of SU-DHL-4 and OCI-Ly18 cells. Finally, imatinib mesylate reduced not only tumor burden in DHL mouse models, but also AID and c-MYC expression in vivo. CONCLUSION: These findings reveal that imatinib mesylate effectively reduces the carcinogenic function of c-MYC in DHL, providing novel strategies for developing therapies targeting c-MYC-driven DHL.

Asunto(s)

RESUMEN

BACKGROUND: Pleural effusion indicates an imbalance between pleural fluid formation and removal. Classified into exudative and transudative, with common symptoms of dry cough, dyspnea and pleuritic chest pain. Confirmed etiology has to be established for effective treatment. OBJECTIVE: Correlate clinical and biochemical profile of various etiologies of pleural effusion. MATERIALS & METHODS: Retrospective observational study of 2 years in the department of respiratory medicine, GMC Bhopal on 280 cases of pleural effusion. RESULTS: Most common etiology was tubercular 202 (72.4%) followed by malignant in 36 (12.8%). With respect to tubercular, malignant pleural effusion has relative risk (RR) of 0.138 (p value < 0.05) in the age group of 51-60 years, which is statistically significant. Patients of tuberculosis complained of fever 158 (78.2%) whereas with malignancy complained of chest pain 16 (44.4%) followed by hemoptysis 12 (33.3%). For hemoptysis, with respect to tubercular, malignant effusion has RR of 5.68 (p value < 0.05) which is significant. History of smoking was significant in malignant effusion with RR of 2.57 (p value < 0.05) as compared to tubercular. Pleural fluid ADA was >70 in 83.7% in tubercular effusion, glucose was <60 mg/dl in 79% tubercular, malignant and bacteriological cause, LDH was >1000 in 88.4% in bacteriological and 72.3% in malignant effusion. CONCLUSION: Lack of tools for confirming diagnosis leads to diagnostic dilemma and delay in treatment initiation, leading to deterioration and untoward fatality in some cases. Our goal is early diagnosis by correlating clinical symptoms with biochemical profile and help initiate rapid treatment.

Asunto(s)

RESUMEN

Phytohormones play a crucial role in regulating growth, productivity, and development while also aiding in the response to diverse environmental changes, encompassing both biotic and abiotic factors. Phytohormone levels in soil and plant tissues are influenced by specific soil bacteria, leading to direct effects on plant growth, development, and stress tolerance. Specific plant growth-promoting bacteria can either synthesize or degrade specific plant phytohormones. Moreover, a wide range of volatile organic compounds synthesized by plant growth-promoting bacteria have been found to influence the expression of phytohormones. Bacteria-plant interactions become more significant under conditions of abiotic stress such as saline soils, drought, and heavy metal pollution. Phytohormones function in a synergistic or antagonistic manner rather than in isolation. The study of plant growth-promoting bacteria involves a range of approaches, such as identifying singular substances or hormones, comparing mutant and non-mutant bacterial strains, screening for individual gene presence, and utilizing omics approaches for analysis. Each approach uncovers the concealed aspects concerning the effects of plant growth-promoting bacteria on plants. Publications that prioritize the comprehensive examination of the private aspects of PGPB and cultivated plant interactions are of utmost significance and crucial for advancing the practical application of microbial biofertilizers. This review explores the potential of PGPB-plant interactions in promoting sustainable agriculture. We summarize the interactions, focusing on the mechanisms through which plant growth-promoting bacteria have a beneficial effect on plant growth and development via phytohormones, with particular emphasis on detecting the synthesis of phytohormones by plant growth-promoting bacteria.

RESUMEN

Background Our study focused on meningitis, an infection that can spread through the bloodstream as a primary or secondary infection from other body parts, such as sinuses, ears, and lungs. It can affect patients who have experienced trauma or surgery, as well as those with congenital defects like spina bifida. Specifically, we examined bacterial, viral, and tuberculous meningitis (TBM) cases. The primary method for confirming the diagnosis of these types of meningitis is to analyze the cerebrospinal fluid (CSF). Early diagnosis can utilize cytological and biochemical parameters. Our objective is to determine CSF's cytological and biochemical profile in patients with these specific types of meningitis. Methods A study was carried out at the central pathology lab from October 24, 2017, to April 24, 2018. CSF samples from suspected meningitis patients were examined for various parameters, including hematological, biochemical, microbiological, and cytomorphological aspects and specific tests for bacterial, fungal, and TBM. The study focused on patients aged 16 and above, excluding those under 16, non-compliant patients, and individuals with specific health conditions. Data were analyzed using IBM SPSS Statistics for Windows, Version 20 (Released 2011; IBM Corp., Armonk, New York, United States), and the results were presented through the use of mean, standard deviation, and percentages. Statistical tests were utilized to compare categorical variables and mean, with a significance level of p<0.05. Results We included a total of 156 cases, with the mean age of presentation being 56.628 years. The male-to-female ratio was 1.0526:1. Of the patients, 81 (52.1%) had been diagnosed with TBM, had elevated adenosine deaminase (ADA) levels of 48.8733±37.43740 IU/L, and CSF lymphocytosis (99%). Additionally, cases of bacterial meningitis showed markedly raised mean total leukocyte count (TLC) of 2085.50±445.47727 cells/mm3 and mean CSF protein levels of 349.45±113.73105 mg/dL. The study found a significant increase in protein levels and a decrease in glucose levels in the CSF of TBM and bacterial meningitis patients compared to those with other causes of meningitis (p<0.001). Guillain-Barre syndrome (GBS) and multiple sclerosis (MS) patients had TLC and ADA within normal limits. CSF ADA level greater than 6 IU/L showed a sensitivity of 97.53% and a specificity of 96.0%, making it the most specific test. A protein level in the CSF greater than 45 mg/dL demonstrated a sensitivity of 98.78% and a specificity of 24.32%, indicating it is sensitive but less specific in diagnosing TBM. Lymphocytic predominance, defined as TLC of more than 5 cells/mm3 with at least 50% of the cells being lymphocytes in the CSF of TBM patients, showed a sensitivity of 97.53% and a specificity of 6.67%. CSF glucose had a sensitivity of 38.27%, making it the least reliable indicator for diagnosing meningitis. Conclusion The CSF analysis is the primary diagnostic method for detecting meningitis. Its cost-effectiveness is a key factor, especially for patients from lower socioeconomic backgrounds in government medical colleges in India, where access to expensive diagnostic tests is limited. The efficiency of CSF analysis for early diagnosing different types of meningitis aids in management, helping to prevent complications and fatal outcomes.

RESUMEN

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

RESUMEN

Introduction and importance: Deficiency of adenosine deaminase 2 (DADA2) is a rare autosomal recessive genetic disorder caused by loss-of-function mutations in the adenosine deaminase 2 (ADA2) gene. This condition primarily manifests in pediatric cases before the age of 10 years, with sporadic cases reported in adults. ADA2 is a critical enzyme involved in macrophage differentiation and immune homeostasis. The clinical manifestations of DADA2 vary widely and can affect multiple organ systems. Our case uniquely highlights an infrequent DADA2 manifestation. Case presentation: An 18-year-old female presented with right flank pain, fever, and a history of joint pain, Raynaud's phenomenon, livedo-like rash, and chronic abdominal pain. Physical examination revealed subcapsular hematoma in the right kidney. Further evaluation showed positive serologic tests for rheumatoid factor and antinuclear antibody (ANA). Genetic testing confirmed DADA2 homozygosity. The patient was discharged on the appropriate medications. Clinical discussion: DADA2 is associated with vascular dysfunction and systemic vasculopathy. The clinical manifestations of DADA2 encompass a spectrum of organ involvement, including the skin, nervous system, gastrointestinal system, renal system, and the cardiovascular system. Early recognition and diagnosis are crucial for appropriate management. Conclusion: This case report highlights the diverse clinical presentations of ADA2 deficiency, specifically focusing on bilateral renal subcapsular hematoma. This finding emphasizes the importance of considering DADA2 as a differential diagnosis in patients presenting with unexplained renal manifestations. Increased awareness of the varied clinical presentations of DADA2 will contribute to earlier diagnosis, appropriate management, and improved outcomes in patients affected by this rare genetic disorder.

RESUMEN

Glioblastoma (GBM), a grade IV brain tumor, presents a severe challenge in treatment and eradication due to its high genetic variability and the existence of stem-like cells with self-renewal potential. Conventional therapies fall short of preventing recurrence and fail to extend the median survival of patients significantly. However, the emergence of gene therapy, which has recently obtained significant clinical outcomes, brings hope. It has the potential to be a suitable strategy for the treatment of GBM. Notably, microRNAs (miRNAs) have been noticed as critical players in the development and progress of GBM. The combined usage of hsa-miR-34a and Cytosine Deaminase (CD) suicide gene and 5-fluorocytosine (5FC) prodrug caused cytotoxicity against U87MG Glioma cells in vitro. The apoptosis and cell cycle arrest rates were measured by flow cytometry. The lentiviral vector generated overexpression of CD/miR-34a in the presence of 5FC significantly promoted apoptosis and caused cell cycle arrest in U87MG cells. The expression level of the BCL2, SOX2, and P53 genes, target genes of hsa-miR-34a, was examined by quantitative real-time PCR. The treatment led to a substantial downregulation of Bcl2 and SOX2 genes while elevating the expression levels of Caspase7 and P53 genes compared to the scrambled control. The hsa-miR-34a hindered the proliferation of GBM cancer cells and elevated apoptosis through the P53-miR-34a-Bcl2 axis. The CD suicide gene with 5FC treatment demonstrated similar results to miR-34a in the apoptosis, cell cycle, and real-time assays. The combination of CD and miR-34a produced a synergistic effect. In vivo, anti-GBM efficacy evaluation in rats bearing intracranial C6 Glioma cells revealed a remarkable induction of apoptosis and a significant inhibition of tumor growth compared with the scrambled control. The simultaneous use of CD/miR-34a with 5FC almost entirely suppressed tumor growth in rat models. The combined application of hsa-miR-34a and CD suicide gene against GBM tumors led to significant induction of apoptosis in U87MG cells and a considerable reduction in tumor growth in vivo.

RESUMEN

BACKGROUND: Deficiency of adenosine deaminase 2 (DADA2) is a complex monogenic disease caused by recessive mutations in the ADA2 gene. DADA2 exhibits a broad clinical spectrum encompassing vasculitis, immunodeficiency, and hematological abnormalities. Yet, the impact of DADA2 on the bone marrow (BM) microenvironment is largely unexplored. OBJECTIVE: This study comprehensively examined the BM and peripheral blood of pediatric and adult patients with DADA2 presenting rheumatologic/immunologic symptoms or severe hematological manifestations. METHODS: Immunophenotyping of hematopoietic stem cells (HSCs), progenitor cells, and mature cell populations was performed for 18 patients with DADA2. We also conducted a characterization of the mesenchymal stromal cells (MSCs). RESULTS: Our study revealed a significant decrease in primitive HSCs and progenitor cells, alongside their reduced clonogenic capacity and multilineage differentiation potential. These BM defects were evident in patients with both severe and non-severe hematological manifestations, including pediatric patients, demonstrating that BM disruption can emerge silently and early on, even in patients who do not show obvious hematological symptoms. Beyond stem cells, there was a reduction in mature cell populations in the BM and peripheral blood, affecting myeloid, erythroid, and lymphoid populations. Furthermore, BM MSCs in DADA2 patients exhibited reduced clonogenic and proliferation capabilities and were more prone to undergo cellular senescence marked by elevated DNA damage. CONCLUSION: Our exploration into the BM landscape of DADA2 patients sheds light on the critical hematological dimension of the disease and emphasizes the importance of vigilant monitoring, even in the case of subclinical presentation.

RESUMEN

This study looked at the possibility of using bacteria that were separated from the rhizosphere of rice plants to promote plant development and offer biological control against pests that affect agriculture. A total of 119 bacteria were isolated from rice rhizospheres collected from six different locations. Of these, 15.47% showed phosphate solubilization, 47.05% showed IAA, 89.07% showed siderophore, and 10.08% showed ACC deaminase activity. Generally, high siderophore production was observed in strains showing ACC deaminase activity. The antagonistic behavior of all strains against the walnut pest Xanthomonas arbiricola was also studied, and eight (6.7%) isolates suppressed the growth of this pathogen (7-43 ± 2 mm zone diameter). It was also noted that these eight isolates showed almost exclusively siderophore activity. In contrast to IAA and siderophore synthesis, the study demonstrated reduced activity levels for phosphate solubilization and ACC deaminase. The 16S rRNA sequence results of some of the bacteria selected in this study and AFLP analysis based on some restriction enzymes showed that the diversity was quite high. According to the 16S rRNA analysis, the high antagonistic effect of strain 71, which is one of the members of the Enterobacter genus, shows that it can be used as a biocontrol agent. In this study, it was revealed in detail that bacteria can be preferred as alternative biological agents for plant growth instead of synthetic fertilizers. This is the first study on this subject in this region, which is one of the important points of the country in terms of rice production. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-024-04077-5.

RESUMEN

A 63-year-old man had been smoking bidis for 25 years and developed tubercular empyema, further complicated by pneumothorax and other pulmonary issues. Over a period of three weeks, the individual experienced a gradual onset of symptoms, including progressive shortness of breath, cough, fever, and chest pain. Radiographic examinations revealed significant left-sided pleural effusion with consolidation and evidence of pneumothorax. Other findings included anemia, hyponatremia, substantially increased lactate dehydrogenase, and adenosine deaminase (ADA), consistent with tubercular or chronic infection. The comprehensive treatment plan involved the administration of antibiotics, antitubercular drugs, draining of the pleural fluid, nebulized bronchodilators, corticosteroids, and broad-spectrum antibiotics. The patient exhibited a positive response, showing notable clinical improvement, which was closely monitored through sequential chest X-rays and ECGs. This would continue to highlight the vital need for early tuberculosis detection in patients with chronic obstructive pulmonary disease due to clinical overlap with other diseases. To diagnose and follow up on tuberculous pleural effusion cases, it was critical to integrate both clinical and radiographic findings with laboratory data. It emphasizes the necessity for a multidisciplinary approach to improve overall treatment outcomes.

RESUMEN

Inborn errors of immunity (IEI) are a diverse group of disorders caused by defects in immune system structure or function, involving both innate and adaptive immunity. The 2022 update of the IEI classification includes 485 distinct disorders, categorized into ten major disease groups. With the rapid development of molecular biology, the specific pathogenesis of many IEI has been revealed, making gene therapy possible in preclinical and clinical research of this type of disease. This article reviews the advancements in gene therapy for IEI, aiming to increase awareness and understanding of these disorders.

Asunto(s)

RESUMEN

S-adenosylmethionine (SAM) is most widely known as the biological methylating agent of methyltransferases and for generation of radicals by the iron-sulfur dependent Radical SAM enzymes. SAM also serves as a substrate in biosynthetic reactions that harvest the aminobutyrate moiety of the methionine, producing methylthioadenosine as a co-product. These reactions are found in the production of polyamines such as spermine, siderophores derived from nicotianamine, and opine metallophores staphylopine and pseudopaline, among others. This procedure defines a highly sensitive, continuous fluorescence assay for the determination of steady state kinetic parameters for enzymes that generate the co-product methylthioadenosine.

Asunto(s)

RESUMEN

BACKGROUND: Deficiency of adenosine deaminase (ADA or ADA1) has broad clinical and genetic heterogeneity. Screening techniques can identify asymptomatic infants whose phenotype and prognosis are indeterminate, and who may carry ADA variants of unknown significance. OBJECTIVE: We systematically assessed the pathogenic potential of rare ADA missense variants to better define the relationship of genotype to red blood cell (RBC) total deoxyadenosine nucleotide (dAXP) content and to phenotype. METHODS: We expressed 46 ADA missense variants in the ADA-deficient SØ3834 strain of Escherichia coli and defined genotype categories (GCs) ranked I to IV by increasing expressed ADA activity. We assessed relationships among GC rank, RBC dAXP, and phenotype in 58 reference patients with 50 different genotypes. We used our GC ranking system to benchmark AlphaMissense for predicting variant pathogenicity, and we used a minigene assay to identify exonic splicing variants in ADA exon 9. RESULTS: The 46 missense variants expressed ∼0.001% to ∼70% of wild-type ADA activity (40% had <0.05% of wild-type ADA activity and 50% expressed >1%). RBC dAXP ranged from undetectable to >75% of total adenine nucleotides and correlated well with phenotype. Both RBC dAXP and clinical severity were inversely related to total ADA activity expressed by both inherited variants. Our GC scoring system performed better than AlphaMissense in assessing variant pathogenicity, particularly for less deleterious variants. CONCLUSION: For ADA deficiency, pathogenicity is a continuum and conditional, depending on the total ADA activity contributed by both inherited variants as indicated by GC rank. However, in patients with indeterminate phenotype identified by screening, RBC dAXP measured at diagnosis may have greater prognostic value than GC rank.

RESUMEN

Introduction Pleural effusion is a challenging diagnosis at times, especially due to the overlap of symptoms in effusions of various etiologies. In this study, we aimed to identify if pleural fluid adenosine deaminase (ADA) or serum C-reactive protein (CRP) can be used as an additional novel biomarker for ADA in diagnosing tubercular, parapneumonic, and malignant pleural effusions. Materials and methods A prospective, observational, cross-sectional study was conducted on 79 patients diagnosed with tubercular, parapneumonic, or malignant pleural effusion from August 2022 to April 2024 at the Department of Respiratory Medicine at Dr. D. Y. Patil Medical College, Hospital & Research Centre, Pimpri, Pune. The pleural fluid ADA/serum CRP ratio was identified in each group, and analysis was done to compare the ratio in each group. The correlation with pleural fluid ADA was also identified. Results A total of 79 patients were enrolled in this study. Out of these patients, 53 (67.1%) were identified as having tubercular pleural effusion, 10 (12.7%) patients had parapneumonic effusion, and 16 (20.3%) had malignant pleural effusion. For malignant effusions, the area under the curve (AUC) using the receiver operating characteristic (ROC) for the ADA/CRP ratio was observed to be 0.862. Sensitivity was 87.50% and specificity was 82.54% at a cut-off value of ≤0.5. The positive predictive value was found to be 56%, and the negative predictive value was found to be 96.3%. For parapneumonic effusions, the AUC using the ROC for the ADA/CRP ratio was observed to be 0.880. Sensitivity was 100% and specificity was 69.57% at a cut-off value of ≤0.67. The positive predictive value was found to be 32.3%, and the negative predictive value was found to be 100%. For tubercular effusions, the AUC using the ROC for the ADA/CRP ratio was observed to be 0.955. Sensitivity was 92.45% and specificity was 88.46% at a cut-off value of >0.54. The positive predictive value was found to be 94.2%, and the negative predictive value was found to be 85.2%. The Pearson correlation coefficient (r) of 0.633 indicates a moderately strong positive linear relationship between ADA and ADA/CRP levels. Conclusion The pleural fluid ADA-to-serum CRP ratio can be used as a useful diagnostic tool for differentiating between tubercular, parapneumonic, and malignant pleural effusions. ADA/CRP ratio has added diagnostic value over ADA. In clinically puzzling scenarios, the ADA/CRP ratio can be a cost-effective tool before opting for a more expensive and invasive procedure, which is also often difficult to obtain in resource-limited healthcare settings. More research with a larger sample size is indicated to incorporate the ADA/CRP ratio as an added diagnostic tool along with ADA.

RESUMEN

The study of plant-microbe interactions is a rapidly growing research field, with increasing attention to the role of seed-borne microbial endophytes in protecting the plant during its development from abiotic and biotic stresses. Recent evidence suggests that seed microbiota is crucial in establishing the plant microbial community, affecting its composition and structure, and influencing plant physiology and ecology. For Theobroma cacao L., the diversity and composition of vertically transmitted microbes have yet to be addressed in detail. We explored the composition and diversity of seed-borne endophytes in cacao pods of commercial genotypes (ICS95, IMC67), recently liberated genotypes from AGROSAVIA (TCS01, TCS19), and landraces from Tumaco (Colombia) (AC9, ROS1, ROS2), to evaluate microbial vertical transmission and establishment in various tissues during plant development. We observed a higher abundance of Pseudomonas and Pantoea genera in the landraces and AGROSAVIA genotypes, while the commercial genotypes presented a higher number of bacteria species but in low abundance. In addition, all the genotypes and plant tissues showed a high percentage of fungi of the genus Penicillium. These results indicate that domestication in cacao has increased bacterial endophyte diversity but has reduced their abundance. We isolated some of these seed-borne endophytes to evaluate their potential as growth promoters and found that Bacillus, Pantoea, and Pseudomonas strains presented high production of indole acetic acid and ACC deaminase activity. Our results suggest that cacao domestication could lead to the loss of essential bacteria for seedling establishment and development. This study improves our understanding of the relationship and interaction between perennial plants and seed-borne microbiota.

Asunto(s)

RESUMEN

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.

RESUMEN

INTRODUCTION: Adenosine deaminase (ADA) in pleural fluid is a useful marker for diagnosing tuberculous pleurisy. However, recent studies have reported a lower specificity of pleural fluid ADA levels. We previously developed a diagnostic flowchart for patients with pleural fluid ADA ≥40 U/L, incorporating variables such as pleural fluid lactate dehydrogenase <825 U/L, predominant pleural fluid neutrophils or cell degeneration, and a pleural fluid ADA/total protein ratio <14. This flowchart was effective in distinguishing between tuberculous pleurisy and other diseases. Here, we conducted a validation analysis of this flowchart. MATERIALS AND METHODS: We retrospectively collected data from 458 patients with pleural fluid ADA concentrations ≥40 U/L across eight institutions from January 2019 to December 2023. The diagnostic accuracy rate, sensitivity, and specificity of the diagnostic flowchart were analysed and compared to those in the original study. RESULTS: Eighty-seven patients were diagnosed with tuberculous pleurisy, and 371 patients were diagnosed with other diseases. The diagnostic accuracy, sensitivity, and specificity for diagnosing tuberculous pleurisy were 77.7%, 86.2%, and 75.7%, respectively. Compared with that in the original study, the rate of tuberculous pleurisy was lower (19.0% vs. 44.5%, p < 0.001), but the diagnostic accuracy rates were not significantly different (p = 0.253). On the basis of the findings from this validation study, we have revised the flowchart to enhance its utility. CONCLUSION: The diagnostic flowchart exhibited high diagnostic accuracy in this validation study, comparable to that in the original study. This validation confirms the effectiveness of the flowchart, even in settings with a low incidence of tuberculosis.

RESUMEN

Deaminase-T7 RNA polymerase fusion (MutaT7) proteins are a growing class of synthetic biology tools used to diversify target genes during in vivo laboratory evolution. To date, MutaT7 chimeras comprise either a deoxyadenosine or deoxycytidine deaminase fused to a T7 RNA polymerase. Their expression drives targeted deoxyadenosine-to-deoxyguanosine or deoxycytidine-to-deoxythymidine mutagenesis, respectively. Here, we repurpose recently engineered substrate-promiscuous general deaminases (GDEs) to establish a substantially simplified system based on a single chimeric enzyme capable of targeting both deoxyadenosine and deoxycytidine. We assess on- and off-target mutagenesis, strand and context preference, and parity of deamination for four different MutaT7GDE constructs. We identify a single chimera that installs all possible transition mutations more efficiently than preexisting, more cumbersome MutaT7 tools. The optimized MutaT7GDE chimera reported herein is a next-generation hypermutator capable of mediating efficient and uniform target-gene diversification during in vivo directed evolution.

Asunto(s)