Multifaceted role for p53 in pancreatic cancer suppression.

The vast majority of human pancreatic ductal adenocarcinomas (PDACs) harbor TP53 mutations, underscoring p53's critical role in PDAC suppression. PDAC can arise when pancreatic acinar cells undergo acinar-to-ductal metaplasia (ADM), giving rise to premalignant pancreatic intraepithelial neoplasias (PanINs), which finally progress to PDAC. The occurrence of TP53 mutations in late-stage PanINs has led to the idea that p53 acts to suppress malignant transformation of PanINs to PDAC. However, the cellular basis for p53 action during PDAC development has not been explored in detail. Here, we leverage a hyperactive p53 variant-p5353,54-which we previously showed is a more robust PDAC suppressor than wild-type p53, to elucidate how p53 acts at the cellular level to dampen PDAC development. Using both inflammation-induced and KRASG12D-driven PDAC models, we find that p5353,54 both limits ADM accumulation and suppresses PanIN cell proliferation and does so more effectively than wild-type p53. Moreover, p5353,54 suppresses KRAS signaling in PanINs and limits effects on the extracellular matrix (ECM) remodeling. While p5353,54 has highlighted these functions, we find that pancreata in wild-type p53 mice similarly show less ADM, as well as reduced PanIN cell proliferation, KRAS signaling, and ECM remodeling relative to Trp53-null mice. We find further that p53 enhances chromatin accessibility at sites controlled by acinar cell identity transcription factors. These findings reveal that p53 acts at multiple stages to suppress PDAC, both by limiting metaplastic transformation of acini and by dampening KRAS signaling in PanINs, thus providing key new understanding of p53 function in PDAC.

Correlation between neuroimaging, neurological phenotype, and functional outcomes in Wilson's disease.

INTRODUCTION: Wilson's disease (WD) is associated with a variety of movement disorders and progressive neurological dysfunction. The aim of this study was to correlate baseline brain magnetic resonance imaging (MRI) features with clinical phenotype and long-term outcomes in chronically treated WD patients. METHODS: Patients were retrospectively selected from an institutional database. Two experienced neuroradiologists reviewed baseline brain MRI. Functional assessment was performed using the World Health Organization Disability Assessment Schedule 2.0 (WHODAS 2.0) scale, and disease severity was classified using the Global Assessment Scale for Wilson's Disease (GASWD). RESULTS: Of 27 patients selected, 14 were female (51.9%), with a mean (standard deviation [SD]) age at onset of 19.5 (7.1) years. Neurological symptoms developed in 22 patients (81.5%), with hyperkinetic symptoms being the most common (70.4%). Baseline brain MRI showed abnormal findings in 18 cases (66.7%), including T2 hyperintensities in 59.3% and atrophy in 29.6%. After a mean (SD) follow-up of 20.9 (11.0) years, WD patients had a mean score of 19.2 (10.2) on WHODAS 2.0 and 6.4 (5.7) on GASWD. The presence of hyperkinetic symptoms correlated with putaminal T2 hyperintensities (p = 0.003), putaminal T2 hypointensities (p = 0.009), and mesencephalic T2 hyperintensities (p = 0.009). Increased functional disability was associated with brain atrophy (p = 0.007), diffusion abnormalities (p = 0.013), and burden of T2 hyperintensities (p = 0.002). A stepwise regression model identified atrophy as a predictor of increased WHODAS 2.0 (p = 0.023) and GASWD (p = 0.007) scores. CONCLUSIONS: Atrophy and, to a lesser extent, deep T2 hyperintensity are associated with functional disability and disease severity in long-term follow-up of WD patients.

Height benefit of GnRH agonists after age 8 in a Portuguese cohort of central precocious puberty.

OBJECTIVE: Idiopathic central precocious puberty (iCPP) is common in paediatric endocrinology. Gonadotropin-releasing hormone agonists (GnRHa) are safe, but the effect on final height and the ideal timing for treatment remains controversial. This study aims to assess the effectiveness of GnRHa on growth outcomes in girls with iCPP treated before and after the age of 8 years old. DESIGN AND PATIENTS: This retrospective longitudinal study evaluated data from Portuguese girls with iCPP who completed treatment between 2010 and 2021. MEASUREMENTS: Auxological and clinical characteristics were compared according to age at treatment onset. RESULTS: A cohort of 134 girls with iCPP, was divided into early treatment (ET) (<8 years, n = 48) and later treatment (LT) groups (≥8 years, n = 86). In both groups, most children presented with Tanner II and III. Tanner IV was more frequent in LT group (p = .003). At the end of treatment, predicted adult height increased in both groups (ET p = .032; LT p = .04) and bone age significantly slowed down in all participants (p = .008, p = .034). The height gain was greater in the ET group, but without significant differences (p = .065). CONCLUSIONS: Treatment with GnRHa improved final height in all girls with iCPP, even when initiated after 8 years. To achieve better outcomes, treatment should be provided promptly after diagnosis.

Adenosine A2A Receptors Contribute to the Radial Migration of Cortical Projection Neurons through the Regulation of Neuronal Polarization and Axon Formation.

Cortical interneurons born in the subpallium reach the cortex through tangential migration, whereas pyramidal cells reach their final position by radial migration. Purinergic signaling via P2Y1 receptors controls the migration of intermediate precursor cells from the ventricular zone to the subventricular zone. It was also reported that the blockade of A2A receptors (A2AR) controls the tangential migration of somatostatin+ interneurons. Here we found that A2AR control radial migration of cortical projection neurons. In A2AR-knockout (KO) mouse embryos or naïve mouse embryos exposed to an A2AR antagonist, we observed an accumulation of early-born migrating neurons in the lower intermediate zone at late embryogenesis. In utero knockdown of A2AR also caused an accumulation of neurons at the lower intermediate zone before birth. This entails the presently identified ability of A2AR to promote multipolar-bipolar transition and axon formation, critical for the transition of migrating neurons from the intermediate zone to the cortical plate. This effect seems to require extracellular ATP-derived adenosine since a similar accumulation of neurons at the lower intermediate zone was observed in mice lacking ecto-5'-nucleotidase (CD73-KO). These findings frame adenosine as a fine-tune regulator of the wiring of cortical inhibitory and excitatory networks.

Unravelling Mechanisms of Doxorubicin-Induced Toxicity in 3D Human Intestinal Organoids.

Doxorubicin is widely used in the treatment of different cancers, and its side effects can be severe in many tissues, including the intestines. Symptoms such as diarrhoea and abdominal pain caused by intestinal inflammation lead to the interruption of chemotherapy. Nevertheless, the molecular mechanisms associated with doxorubicin intestinal toxicity have been poorly explored. This study aims to investigate such mechanisms by exposing 3D small intestine and colon organoids to doxorubicin and to evaluate transcriptomic responses in relation to viability and apoptosis as physiological endpoints. The in vitro concentrations and dosing regimens of doxorubicin were selected based on physiologically based pharmacokinetic model simulations of treatment regimens recommended for cancer patients. Cytotoxicity and cell morphology were evaluated as well as gene expression and biological pathways affected by doxorubicin. In both types of organoids, cell cycle, the p53 signalling pathway, and oxidative stress were the most affected pathways. However, significant differences between colon and SI organoids were evident, particularly in essential metabolic pathways. Short time-series expression miner was used to further explore temporal changes in gene profiles, which identified distinct tissue responses. Finally, in silico proteomics revealed important proteins involved in doxorubicin metabolism and cellular processes that were in line with the transcriptomic responses, including cell cycle and senescence, transport of molecules, and mitochondria impairment. This study provides new insight into doxorubicin-induced effects on the gene expression levels in the intestines. Currently, we are exploring the potential use of these data in establishing quantitative systems toxicology models for the prediction of drug-induced gastrointestinal toxicity.

A Transcriptomic Approach to Elucidate the Mechanisms of Gefitinib-Induced Toxicity in Healthy Human Intestinal Organoids.

Gefitinib is a tyrosine kinase inhibitor (TKI) that selectively inhibits the epidermal growth factor receptor (EGFR), hampering cell growth and proliferation. Due to its action, gefitinib has been used in the treatment of cancers that present abnormally increased expression of EGFR. However, side effects from gefitinib therapy may occur, among which diarrhoea is most common, that can lead to interruption of the planned therapy in the more severe cases. The mechanisms underlying intestinal toxicity induced by gefitinib are not well understood. Therefore, this study aims at providing insight into these mechanisms based on transcriptomic responses induced in vitro. A 3D culture of healthy human colon and small intestine (SI) organoids was exposed to 0.1, 1, 10 and 30 µM of gefitinib, for a maximum of three days. These drug concentrations were selected using physiologically-based pharmacokinetic simulation considering patient dosing regimens. Samples were used for the analysis of viability and caspase 3/7 activation, image-based analysis of structural changes, as well as RNA isolation and sequencing via high-throughput techniques. Differential gene expression analysis showed that gefitinib perturbed signal transduction pathways, apoptosis, cell cycle, FOXO-mediated transcription, p53 signalling pathway, and metabolic pathways. Remarkably, opposite expression patterns of genes associated with metabolism of lipids and cholesterol biosynthesis were observed in colon versus SI organoids in response to gefitinib. These differences in the organoids' responses could be linked to increased activated protein kinase (AMPK) activity in colon, which can influence the sensitivity of the colon to the drug. Therefore, this study sheds light on how gefitinib induces toxicity in intestinal organoids and provides an avenue towards the development of a potential tool for drug screening and development.

Foliar application of 3-hydroxy-4-pyridinone Fe-chelate [Fe(mpp)3 ] induces responses at the root level amending iron deficiency chlorosis in soybean.

Iron (Fe) deficiency chlorosis (IDC) affects the growth of several crops, especially when growing in alkaline soils. The application of synthetic Fe-chelates is one of the most commonly used strategies in IDC amendment, despite their associated negative environmental impacts. In a previous work, the Fe-chelate tris(3-hydroxy-1-(H)-2-methyl-4-pyridinonate) iron(III) [Fe(mpp)3 ] has shown great potential for alleviating IDC in soybean (Glycine max) in the early stages of plant development under hydroponic conditions. Herein, its efficacy was verified under soil conditions in soybean grown from seed to full maturity. Chlorophyll levels, plant growth, root and shoot mineral accumulation (K, Mg, Ca, Na, P, Mn, Zn, Ni, and Co) and FERRITIN expression were accessed at V5 phenological stage. Compared to a commonly used Fe chelate, FeEDDHA, supplementation with [Fe(mpp)3 ] led to a 29% higher relative chlorophyll content, 32% higher root biomass, 36% higher trifoliate Fe concentration, and a twofold increase in leaf FERRITIN gene expression. [Fe(mpp)3 ] supplementation also resulted in increased accumulation of P, K, Zn, and Co. At full maturity, the remaining plants were harvested and [Fe(mpp)3 ] application led to a 32% seed yield increase when compared to FeEDDHA. This is the first report on the use of [Fe(mpp)3 ] under alkaline soil conditions for IDC correction, and we show that its foliar application has a longer-lasting effect than FeEDDHA, induces efficient root responses, and promotes the uptake of other nutrients.

New insights into the mechanisms underlying 5-fluorouracil-induced intestinal toxicity based on transcriptomic and metabolomic responses in human intestinal organoids.

5-Fluorouracil (5-FU) is a widely used chemotherapeutical that induces acute toxicity in the small and large intestine of patients. Symptoms can be severe and lead to the interruption of cancer treatments. However, there is limited understanding of the molecular mechanisms underlying 5-FU-induced intestinal toxicity. In this study, well-established 3D organoid models of human colon and small intestine (SI) were used to characterize 5-FU transcriptomic and metabolomic responses. Clinically relevant 5-FU concentrations for in vitro testing in organoids were established using physiologically based pharmacokinetic simulation of dosing regimens recommended for cancer patients, resulting in exposures to 10, 100 and 1000 µM. After treatment, different measurements were performed: cell viability and apoptosis; image analysis of cell morphological changes; RNA sequencing; and metabolome analysis of supernatant from organoids cultures. Based on analysis of the differentially expressed genes, the most prominent molecular pathways affected by 5-FU included cell cycle, p53 signalling, mitochondrial ATP synthesis and apoptosis. Short time-series expression miner demonstrated tissue-specific mechanisms affected by 5-FU, namely biosynthesis and transport of small molecules, and mRNA translation for colon; cell signalling mediated by Rho GTPases and fork-head box transcription factors for SI. Metabolomic analysis showed that in addition to the effects on TCA cycle and oxidative stress in both organoids, tissue-specific metabolic alterations were also induced by 5-FU. Multi-omics integration identified transcription factor E2F1, a regulator of cell cycle and apoptosis, as the best key node across all samples. These results provide new insights into 5-FU toxicity mechanisms and underline the relevance of human organoid models in the safety assessment in drug development.

Nanoaggregate-forming lipid-conjugated AS1411 aptamer as a promising tumor-targeted delivery system of anticancer agents in vitro.

Nanoparticles offer targeted delivery of drugs with minimal toxicity to surrounding healthy tissue and have great potential in the management of human papillomavirus (HPV)-related diseases. We synthesized lipid-modified AS1411 aptamers capable of forming nanoaggregates in solution containing Mg2+. The nanoaggregates presented suitable properties for pharmaceutical applications such as small size (100 nm), negative charge, and drug release. The nanoaggregates were loaded with acridine orange derivative C8 for its specific delivery into cervical cancer cell lines and HPV-positive tissue biopsies. This improved inhibition of HeLa proliferation and cell uptake without significantly affecting healthy cells. Finally, the nanoaggregates were incorporated in a gel formulation with promising tissue retention properties aiming at developing a local delivery strategy of the nanoaggregates in the female genital tract. Collectively, these findings suggest that the nanoformulation protocol has great potential for the delivery of both anticancer and antiviral agents, becoming a novel modality for cervical cancer management.

Quantitative Systems Toxicology Modeling To Address Key Safety Questions in Drug Development: A Focus of the TransQST Consortium.

Omics data have been increasingly generated with limited demonstrated value in drug safety assessment. The TransQST consortium was launched to use omics and other data in mechanistic-based quantitative systems toxicology (QST) models to evaluate their potential use in species translation.

Metabolic engineering strategies for butanol production in Escherichia coli.

The global market of butanol is increasing due to its growing applications as solvent, flavoring agent, and chemical precursor of several other compounds. Recently, the superior properties of n-butanol as a biofuel over ethanol have stimulated even more interest. (Bio)butanol is natively produced together with ethanol and acetone by Clostridium species through acetone-butanol-ethanol fermentation, at noncompetitive, low titers compared to petrochemical production. Different butanol production pathways have been expressed in Escherichia coli, a more accessible host compared to Clostridium species, to improve butanol titers and rates. The bioproduction of butanol is here reviewed from a historical and theoretical perspective. All tested rational metabolic engineering strategies in E. coli to increase butanol titers are reviewed: manipulation of central carbon metabolism, elimination of competing pathways, cofactor balancing, development of new pathways, expression of homologous enzymes, consumption of different substrates, and molecular biology strategies. The progress in the field of metabolic modeling and pathway generation algorithms and their potential application to butanol production are also summarized here. The main goals are to gather all the strategies, evaluate the respective progress obtained, identify, and exploit the outstanding challenges.

Carbon monoxide released by CORM-A1 prevents yeast cell death via autophagy stimulation.

Autophagy is an autodigestive process, promoting cytoprotection by the elimination of dysfunctional organelles, misfolded proteins and toxic aggregates. Carbon monoxide (CO) is an endogenous gasotransmitter that under low concentrations prevents cell death and inflammation. For the first time, the role of autophagy in CO-mediated cytoprotection against oxidative stress was evaluated in the model yeast Saccharomyces cerevisiae. The boron-based CO-releasing molecule, CORM-A1, was used to deliver CO. CORM-A1 partially prevented oxidative stress-induced cell death in yeast. Likewise, CORM-A1 activated autophagy under basal physiological conditions, which were assessed by autophagic flux and the expression of mCherry-Atg8 or GFP-Atg8. Inhibition of autophagy by knocking out key autophagic genes in yeast (ATG8 or ATG11) blocked CORM-A1 cytoprotective effect, indicating the critical role of autophagy in CO-induced cytoprotection. The CO-mediated cytoprotection via autophagy induction observed in yeast was validated in primary cultures of astrocytes, a well-characterized model for CO's cytoprotective functions. As in yeast, CORM-A1 prevented oxidative stress-induced cell death in an autophagy-dependent manner in astrocytes. Overall, our data support the cytoprotective action of CO against oxidative stress. CO promotes cytoprotection in yeast via autophagy, opening new possibilities for the study of molecular mechanisms of CO's biological functions using this powerful eukaryotic model.

Treatment modalities and outcomes in pediatric Cushing's disease - report of three cases and literature review.

OBJECTIVES: Cushing's disease (CD) is a diagnostic and therapeutic challenge, especially in pediatric patients. CD, primarily caused by adrenocorticotropic hormone-secreting pituitary adenomas, manifests typically with growth retardation and weight gain. There are no published guidelines for pediatric patients. CASE PRESENTATION: We report three pediatric patients diagnosed with CD in a Portuguese tertiary hospital. All patients presented with hypercortisolism features. All patients underwent transsphenoidal pituitary surgery (TSS) as a first-choice treatment; however, it was unsuccessful in one patient and the other patients experienced recurrence. Patients were submitted to different approaches so basal serum cortisol levels could be achieved. Two of three patients achieved remission. CONCLUSIONS: TSS remains the first-line treatment yet challenging due to microadenomas and technical complexities. Medical therapy with agents like metyrapone or ketoconazole, pituitary radiotherapy, or bilateral adrenalectomy are, usually, second-line interventions, unless there is a contraindication to surgery. Our findings support the finding that a shorter hypothalamic-pituitary-adrenal axis recovery time increases the risk of recurrence of CD. Our cases illustrate the intricate management and variable outcomes of pediatric CD, underscoring the importance of multidisciplinary care and continuous surveillance.

Olmesartan-Induced Enteropathy: When the Treatment of One Disease Causes Another.

Olmesartan is an angiotensin II receptor antagonist used for the management of hypertension. This drug can lead to an enteropathy that clinically and histologically resembles coeliac disease. Symptoms may appear months or years after the introduction of the drug and usually resolve after discontinuation. The authors present a case of an 86-year-old woman with hypertension who was treated with olmesartan for 10 years. She presented to the emergency department with diarrhoea after three months of development and weight loss. The aetiological study that was conducted excluded infectious, inflammatory, endocrinological, and neoplastic causes. The pathological anatomy of the duodenal biopsy was suggestive of coeliac disease, but the serology was not compatible. The patient presented complete remission of the condition with the suspension of the drug and subsequent recrudescence when, by self-initiation, she resumed olmesartan. This case study aims to alert readers of a rare cause of enteropathy with a clinical manifestation that mimics coeliac disease. Olmesartan-induced enteropathy seems to be a diagnosis of exclusion and should be considered in patients chronically medicated with olmesartan.

Nonalcoholic Fatty Liver Disease and Continuous Metabolic Syndrome in Adolescents with Overweight/Obesity.

INTRODUCTION: Nonalcoholic fatty liver disease is the leading cause of pediatric chronic liver disease. Although nonalcoholic fatty liver disease is closely associated with obesity, its relationship with metabolic syndrome in children is not fully understood. The main aim of this study was to evaluate the association between nonalcoholic fatty liver disease and a combination of cardiometabolic risk factors in adolescents with overweight/obesity, using a pediatric metabolic syndrome score (PsiMS) to predict metabolic syndrome. METHODS: A retrospective cohort study was conducted. Subjects with overweight/obesity aged 10 to 17 followed at two clinical centers in Portugal (2018 - 2021) were enrolled. The independent association of nonalcoholic fatty liver disease with PsiMS, and of other potential predictors, was tested through multiple regression analyses. Receiver operator characteristic curve analysis was performed to estimate the optimal cutoff of PsiMS to discriminate metabolic syndrome. RESULTS: Eighty-four subjects were included (median age at baseline 11.5 years). The prevalence rate of nonalcoholic fatty liver disease was 51% and the prevalence rate of metabolic syndrome was 7%. The mean PsiMS was 2.05 ± 0.48 at the first evaluation, and 2.11 ± 0.52 at the last evaluation (mean follow-up time was 15 months). The nonalcoholic fatty liver disease group had significantly (p < 0.05) higher weight and body mass index z-scores, higher rate of severe obesity and higher waist circumference percentile. PsiMS was highly accurate in predicting metabolic syndrome (area under the curve = 0.96), with an optimal cutoff of 2.46 (sensitivity 100%, specificity 89%). In the univariate analysis, no statistically significant association was observed between nonalcoholic fatty liver disease and PsiMS. In the multiple regression analysis, female sex had a negative association with PsiMS (first and last evaluation). Independent predictors of a higher PsiMS at first evaluation were: ≥ 2 metabolic syndrome criteria, body mass index z-score, insulin resistance and dyslipidemia. At the last evaluation, independent predictors of a higher PsiMS were: nonalcoholic fatty liver disease, baseline PsiMS and body mass index increase from baseline. CONCLUSION: The results suggest a good performance of the PsiMS to assess metabolic syndrome and that nonalcoholic fatty liver disease is associated with PsiMS at follow-up.

Chronic Lithium Intoxication: A Challenging Diagnosis.

Lithium has been used in clinical practice since the 1970s. This medication is commonly used to treat and prevent bipolar disorder, but it has a narrow therapeutic index, making toxicity a frequent occurrence. Chronic lithium intoxication can arise due to progressive accumulation, particularly in contexts of dehydration. The effects of chronic lithium intoxication on the nervous, renal, and cardiac systems, as well as on the thyroid and parathyroid glands, are well documented in the literature. The authors present the case of a 66-year-old woman with schizoaffective psychosis and chronic kidney disease, admitted due to altered mental status and dysarthria. Notwithstanding an earlier clinical recommendation to cease lithium administration more than a year ago, the patient continued its usage, culminating in neurological, cardiac, renal, and endocrine manifestations. Although the diagnosis was delayed, her clinical progression was favorable, obviating the need for renal replacement therapy. This case highlights the importance of a detailed medical history and the diagnostic challenges in clinical practice. The use of this drug without proper monitoring can lead to multisystem dysfunction.