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Aberrant gene expression patterns in acute myeloid leukemia (AML) with balanced chromosomal translocations are often associated with dysregulation of epigenetic modifiers. The AML1/ETO (RUNX1/MTG8) fusion protein, caused by the translocation (8;21)(q22;q22), leads to the epigenetic repression of its target genes. We aimed in this work to identify critical epigenetic modifiers, on which AML1/ETO-positive AML cells depend on for proliferation and survival using shRNA library screens and global transcriptomics approaches. Using shRNA library screens, we identified 41 commonly depleted genes in two AML1/ETO-positive cell lines Kasumi-1 and SKNO-1. We validated, genetically and pharmacologically, DNMT1 and ATR using several AML1/ETO-positive and negative cell lines. We also demonstrated in vivo differentiation of myeloblasts after treatment with the DNMT1 inhibitor decitabine in a patient with an AML1/ETO-positive AML. Bioinformatic analysis of global transcriptomics after AML1/ETO induction in 9/14/18-U937 cells identified 973 differentially expressed genes (DEGs). Three genes (PARP2, PRKCD, and SMARCA4) were both downregulated after AML1/ETO induction, and identified in shRNA screens. In conclusion, using unbiased shRNA library screens and global transcriptomics, we have identified several driver epigenetic regulators for proliferation in AML1/ETO-positive AML. DNMT1 and ATR were validated and are susceptible to pharmacological inhibition by small molecules showing promising preclinical and clinical efficacy.
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Monogenic kidney diseases result from an abundance of potential genes carrying pathogenic variants. These conditions are primarily recognized for manifesting as kidney disorders, defined as an impairment of the structure and/or function of the kidneys. However, the impact of these genetic disorders extends far beyond the kidneys, giving rise to a diverse spectrum of extrarenal manifestations. These manifestations can affect any organ system throughout the body, leading to a complex clinical presentation that demands a comprehensive understanding and interdisciplinary management of affected persons. The intricate interplay between genetic variants, molecular pathways, and systemic interactions underscores the importance of exploring the extrarenal aspects of inherited kidney diseases. This exploration not only deepens our comprehension of the diseases themselves but also opens avenues for more holistic diagnostics, treatment strategies, and improved interdisciplinary patient care. This article delves into the intricate realm of extrarenal manifestations in inherited kidney diseases, shedding light on the far-reaching impacts that these genetic conditions can exert beyond the confines of the kidney system.
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This meta-research study aims to evaluate the agreement of effect estimates between bodies of evidence (BoE) from RCTs and cohort studies included in the same nutrition evidence synthesis, to identify factors associated with disagreement, and to replicate the findings of a previous study. We searched Medline, Epistemonikos and the Cochrane Database of Systematic Reviews for nutrition systematic reviews that included both RCTs and cohort studies for the same patient-relevant outcome or intermediate-disease marker. We rated similarity of PI/ECO (population, intervention/exposure, comparison, outcome) between BoE from RCTs and cohort studies. Agreement of effect estimates across BoE was analysed by pooling ratio of risk ratios (RRR) for binary outcomes and difference of standardised mean differences (DSMD) for continuous outcomes. We performed subgroup and sensitivity analyses to explore determinants associated with disagreements. We included 82 BoE-pairs from 51 systematic reviews. For binary outcomes, the RRR was 1.04 (95% confidence interval (CI) 0.99 to 1.10, I2 = 59%, τ2 = 0.02, prediction interval (PI) 0.77 to 1.41). For continuous outcomes, the pooled DSMD was - 0.09 (95% CI - 0.26 to 0.09, PI - 0.55 to 0.38). Subgroup analyses yielded that differences in type of intake/exposure were drivers towards disagreement. We replicated the findings of a previous study, where on average RCTs and cohort studies had similar effect estimates. Disagreement and wide prediction intervals were mainly driven by PI/ECO-dissimilarities. More research is needed to explore other potentially influencing factors (e.g. risk of bias) on the disagreement between effect estimates of both BoE.Trial registration: CRD42021278908.
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Ensayos Clínicos Controlados Aleatorios como Asunto , Humanos , Estudios de CohortesRESUMEN
BACKGROUND: There is growing evidence that substituting animal-based with plant-based foods is associated with a lower risk of cardiovascular diseases (CVD), type 2 diabetes (T2D), and all-cause mortality. Our aim was to summarize and evaluate the evidence for the substitution of any animal-based foods with plant-based foods on cardiometabolic health and all-cause mortality in a systematic review and meta-analysis. METHODS: We systematically searched MEDLINE, Embase, and Web of Science to March 2023 for prospective studies investigating the substitution of animal-based with plant-based foods on CVD, T2D, and all-cause mortality. We calculated summary hazard ratios (SHRs) and 95% confidence intervals (95% CI) using random-effects meta-analyses. We assessed the certainty of evidence (CoE) using the GRADE approach. RESULTS: In total, 37 publications based on 24 cohorts were included. There was moderate CoE for a lower risk of CVD when substituting processed meat with nuts [SHR (95% CI): 0.73 (0.59, 0.91), n = 8 cohorts], legumes [0.77 (0.68, 0.87), n = 8], and whole grains [0.64 (0.54, 0.75), n = 7], as well as eggs with nuts [0.83 (0.78, 0.89), n = 8] and butter with olive oil [0.96 (0.95, 0.98), n = 3]. Furthermore, we found moderate CoE for an inverse association with T2D incidence when substituting red meat with whole grains/cereals [0.90 (0.84, 0.96), n = 6] and red meat or processed meat with nuts [0.92 (0.90, 0.94), n = 6 or 0.78 (0.69, 0.88), n = 6], as well as for replacing poultry with whole grains [0.87 (0.83, 0.90), n = 2] and eggs with nuts or whole grains [0.82 (0.79, 0.86), n = 2 or 0.79 (0.76, 0.83), n = 2]. Moreover, replacing red meat for nuts [0.93 (0.91, 0.95), n = 9] and whole grains [0.96 (0.95, 0.98), n = 3], processed meat with nuts [0.79 (0.71, 0.88), n = 9] and legumes [0.91 (0.85, 0.98), n = 9], dairy with nuts [0.94 (0.91, 0.97), n = 3], and eggs with nuts [0.85 (0.82, 0.89), n = 8] and legumes [0.90 (0.89, 0.91), n = 7] was associated with a reduced risk of all-cause mortality. CONCLUSIONS: Our findings indicate that a shift from animal-based (e.g., red and processed meat, eggs, dairy, poultry, butter) to plant-based (e.g., nuts, legumes, whole grains, olive oil) foods is beneficially associated with cardiometabolic health and all-cause mortality.
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Enfermedades Cardiovasculares , Diabetes Mellitus Tipo 2 , Humanos , Animales , Dieta , Estudios Prospectivos , Diabetes Mellitus Tipo 2/epidemiología , Aceite de Oliva , Verduras , Carne , Enfermedades Cardiovasculares/epidemiología , Enfermedades Cardiovasculares/prevención & control , Enfermedades Cardiovasculares/complicaciones , Mantequilla , Factores de RiesgoRESUMEN
BACKGROUND: According to current evidence, every 10th to 11th adult with chronic kidney disease (CKD) has a monogenic disease of the kidney. METHODS: This review is based on reported studies in which molecular genetic diagnostic techniques were used to investigate monogenic kidney diseases in adults with CKD. The studies were identified by a selective literature search using predefined criteria. RESULTS: In 12 selected studies, diagnostic variants of 179 different genes were identified in 1467 out of 6607 study participants with CKD (22.2%). More than 60% of these variants affected 8 genes (PKD1, PKD2, COL4A3, COL4A4, COL4A5, UMOD, MUC1, HNF1B). Three diseases are associated with these genes: autosomal dominant polycystic kidney disease (ADPKD), Alport syndrome, and autosomal dominant tubulo-interstitial kidney disease (ADTKD). Physicians treating patients with CKD should be alert to the presence of any red flags, such as onset at a young age, a positive family history, or hematuria of unknown cause. When a genetic etiology is suspected, a specialized work-up is indicated, often including a molecular genetic investigation. A positive genetic finding usually leads to a modification of the patient's specific diagnosis and/or treatment. CONCLUSION: Awareness of the high prevalence of monogenic kidney diseases in adults with CKD and alertness to their suggestive clinical features are crucial for the timely initiation of targeted diagnostic testing. The molecular genetic identification of these diseases is a prerequisite for appropriate patient management.
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OBJECTIVES: Our aim was to investigate if and how Cochrane nutrition reviews assess dietary adherence to a specific dietary regimen. STUDY DESIGN AND SETTING: Cochrane nutrition reviews fulfilling the following criteria were included: systematic review of randomized controlled trials including adults and investigating the effect of caloric restriction, dietary pattern, foods, nutrients, supplements, or other nutrition-related-interventions. Extensive data extraction and descriptive statistics were conducted. RESULTS: Overall, 226 Cochrane reviews were included. Most reviews mentioned dietary adherence in the main text (n = 174), predominantly in the Methods and Results. Dietary adherence was assessed in 76 reviews and defined in 19. It was included in the risk of bias (RoB) assessment in 20 reviews with nine using a newly created RoB domain for dietary adherence, and considered as outcome in 37 reviews. Seventy-five reviews addressed degree of adherence and five treatment effects considering the degree of adherence. CONCLUSION: Dietary adherence was reported in a heterogeneous manner in Cochrane nutrition reviews. Due to its high importance, we suggest that systematic reviews report the assessment and degree of dietary adherence measured in primary studies. Dietary adherence can further be examined as outcome, evaluated within the RoB (deviations from intended interventions) and included in sensitivity analyses.
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Dieta , Adulto , Humanos , Sesgo , Medición de Riesgo , Revisiones Sistemáticas como Asunto , Ensayos Clínicos Controlados Aleatorios como AsuntoRESUMEN
OBJECTIVES: This study aimed to identify and describe the use of the Grading of Recommendations Assessment, Development and Evaluation (GRADE) dose-response gradient domain to upgrade the certainty of evidence (CoE) in nutrition systematic reviews (SRs). STUDY DESIGN AND SETTING: We searched for SRs of observational studies of nutrition topics that used GRADE and upgraded the CoE of at least one outcome for a dose-response gradient or reported reasons for not upgrading. RESULTS: Within eligible SRs (21/281), 123 of 371 outcomes were upgraded for a dose-response gradient. For 118 outcomes, the authors conducted linear dose-response analyses, and for 106 outcomes, the authors conducted nonlinear dose-response analyses. From these, 107 outcomes showed a statistically significant (P < 0.05) association in the linear dose-response model, and for 28 outcomes, the test for nonlinearity was statistically significant. The CoE for 0.8% of all outcomes was rated as high, 47.2% as moderate, 43.9% as low, and 8.1% as very low. Fifty-five percent of outcomes that were upgraded for a dose-response gradient were already downgraded for at least one domain. This is contrary to GRADE guidance. CONCLUSION: The approach for rating up the CoE for dose-response relationship is not consistent in nutrition reviews, likely because of a lack of clear guidance for when and how to do it. Therefore, more comprehensive GRADE guidance is necessary to enhance the correct use and comparability of dose-response upgrading.
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Multiprotein complexes control the behavior of cells, such as of lymphocytes of the immune system. Methods to affinity purify protein complexes and to determine their interactome by mass spectrometry are thus widely used. One drawback of these methods is the presence of false positives. In fact, the elution of the protein of interest (POI) is achieved by changing the biochemical properties of the buffer, so that unspecifically bound proteins (the false positives) may also elute. Here, we developed an optogenetics-derived and light-controlled affinity purification method based on the light-regulated reversible protein interaction between phytochrome B (PhyB) and its phytochrome interacting factor 6 (PIF6). We engineered a truncated variant of PIF6 comprising only 22 amino acids that can be genetically fused to the POI as an affinity tag. Thereby the POI can be purified with PhyB-functionalized resin material using 660 nm light for binding and washing, and 740 nm light for elution. Far-red light-induced elution is effective but very mild as the same buffer is used for the wash and elution. As proof-of-concept, we expressed PIF-tagged variants of the tyrosine kinase ZAP70 in ZAP70-deficient Jurkat T cells, purified ZAP70 and associating proteins using our light-controlled system, and identified the interaction partners by quantitative mass spectrometry. Using unstimulated T cells, we were able to detect the known interaction partners, and could filter out all other proteins.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Luz , Péptidos/metabolismo , Fitocromo B/metabolismo , Proteína Tirosina Quinasa ZAP-70/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/química , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Unión Competitiva/efectos de la radiación , Cromatografía Liquida/métodos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Células Jurkat , Optogenética/métodos , Péptidos/genética , Fitocromo B/genética , Unión Proteica/efectos de la radiación , Espectrometría de Masas en Tándem/métodos , Proteína Tirosina Quinasa ZAP-70/genéticaRESUMEN
The detection of antibodies from blood sera is crucial for diagnostic purposes. Miniaturized protein assays in combination with microfluidic setups hold great potential by enabling automated handling and multiplexed analyses. Yet, the separate expression, purification, and storage of many individual proteins are time consuming and limit applicability. In vitro cell-free expression has been proposed as an alternative procedure for the generation of protein assays. We report the successful in vitro expression of different model proteins from DNA templates with an optimized expression mix. His10-tagged proteins were specifically captured and immobilized on a Ni-NTA coated sensor surface directly from the in vitro expression mix. Finally, the specific binding of antibodies from rabbit-derived blood sera to the immobilized proteins was monitored by imaging reflectometric interferometry (iRIf). Antibodies in the blood sera could be identified by binding to the respective epitopes with minimal cross reactivity. The results show the potential of in vitro expression and label-free detection for binding assays in general and diagnostic purposes in specific.