Correction: Patient-derived oral mucosa organoids as an in vitro model for methotrexate induced toxicity in pediatric acute lymphoblastic leukemia.

[This corrects the article DOI: 10.1371/journal.pone.0231588.].

Patient-derived oral mucosa organoids as an in vitro model for methotrexate induced toxicity in pediatric acute lymphoblastic leukemia.

We have recently established a protocol to grow wildtype human oral mucosa organoids. These three-dimensional structures can be maintained in culture long-term, do not require immortalization, and recapitulate the multilayered composition of the epithelial lining of the oral mucosa. Here, we validate the use of this model to study the effect of Leucovorin (LV) on Methotrexate (MTX)-induced toxicity. MTX is a chemotherapeutic agent used in the treatment of pediatric acute lymphoblastic leukemia. Although effective, the use of MTX often results in severe side-effects, including oral mucositis, which is characterized by epithelial cell death. Here, we show that organoids are sensitive to MTX, and that the addition of LV reduces MTX toxicity, in both a concentration- and timing-dependent manner. Additionally, we show that a 24 hour 'pretreatment' with LV reduces MTX-induced cell death, suggesting that such a pretreatment could decrease mucositis in patients. Taken together, we provide the first in vitro model to study the effect of MTX on wildtype oral mucosa cells. Our findings underscore the relevance of the clinically applied LV regimen and highlight the potential of this model to further optimize modifications in dosing and timing of Leucovorin on oral mucosa cells.

Role of proton-coupled folate transporter in pemetrexed resistance of mesothelioma: clinical evidence and new pharmacological tools.

BACKGROUND: Thymidylate synthase (TS) has a predictive role in pemetrexed treatment of mesothelioma; however, additional chemoresistance mechanisms are poorly understood. Here, we explored the role of the reduced-folate carrier (RFC/SLC19A1) and proton-coupled folate transporter (PCFT/SLC46A1) in antifolate resistance in mesothelioma. PATIENTS AND METHODS: PCFT, RFC and TS RNA and PCFT protein levels were determined by quantitative RT-PCR of frozen tissues and immunohistochemistry of tissue-microarrays, respectively, in two cohorts of pemetrexed-treated patients. Data were analyzed by t-test, Fisher's/log-rank test and Cox proportional models. The contribution of PCFT expression and PCFT-promoter methylation to pemetrexed activity were evaluated in mesothelioma cells and spheroids, through 5-aza-2'-deoxycytidine-mediated demethylation and siRNA-knockdown. RESULTS: Pemetrexed-treated patients with low PCFT had significantly lower rates of disease control, and shorter overall survival (OS), in both the test (N = 73, 11.3 versus 20.1 months, P = 0.01) and validation (N = 51, 12.6 versus 30.3 months, P = 0.02) cohorts. Multivariate analysis confirmed PCFT-independent prognostic role. Low-PCFT protein levels were also associated with shorter OS. Patients with both low-PCFT and high-TS levels had the worst prognosis (OS, 5.5 months), whereas associations were neither found for RFC nor in pemetrexed-untreated patients. PCFT silencing reduced pemetrexed sensitivity, whereas 5-aza-2'-deoxycytidine overcame resistance. CONCLUSIONS: These findings identify for the first time PCFT as a novel mesothelioma prognostic biomarker, prompting prospective trials for its validation. Moreover, preclinical data suggest that targeting PCFT-promoter methylation might eradicate pemetrexed-resistant cells characterized by low-PCFT expression.

Vitamin B1 and B6 in the malaria parasite: requisite or dispensable?

Vitamins are essential compounds mainly involved in acting as enzyme co-factors or in response to oxidative stress. In the last two years it became apparent that apicomplexan parasites are able to generate B vitamers such as vitamin B1 and B6 de novo. The biosynthesis pathways responsible for vitamin generation are considered as drug targets, since both provide a high degree of selectivity due to their absence in the human host. This report updates the current knowledge about vitamin B1 and B6 biosynthesis in malaria and other apicomplexan parasites. Owing to the urgent need for novel antimalarials, the significance of the biosynthesis and salvage of these vitamins is critically discussed in terms of parasite survival and their exploitation for drug development.

Vitamin B1 and B6 in the malaria parasite: requisite or dispensable?

Vitamins are essential compounds mainly involved in acting as enzyme co-factors or in response to oxidative stress. In the last two years it became apparent that apicomplexan parasites are able to generate B vitamers such as vitamin B1 and B6 de novo. The biosynthesis pathways responsible for vitamin generation are considered as drug targets, since both provide a high degree of selectivity due to their absence in the human host. This report updates the current knowledge about vitamin B1 and B6 biosynthesis in malaria and other apicomplexan parasites. Owing to the urgent need for novel antimalarials, the significance of the biosynthesis and salvage of these vitamins is critically discussed in terms of parasite survival and their exploitation for drug development.

Neurodegeneration and chronic renal failure in methylmalonic aciduria--a pathophysiological approach.

In the last decades the survival of patients with methylmalonic aciduria has been improved. However, the overall outcome of affected patients remains disappointing. The disease course is often complicated by acute life-threatening metabolic crises, which can result in multiple organ failure or even death, resembling primary defects of mitochondrial energy metabolism. Biochemical abnormalities during metabolic derangement, such as metabolic acidosis, ketonaemia/ketonuria, lactic acidosis, hypoglycaemia and hyperammonaemia, suggest mitochondrial dysfunction. In addition, long-term complications such as chronic renal failure and neurological disease are frequently found. Neuropathophysiological studies have focused on various effects caused by accumulation of putatively toxic organic acids, the so-called 'toxic metabolite' hypothesis. In previous studies, methylmalonate (MMA) has been considered as the major neurotoxin in methylmalonic aciduria, whereas more recent studies have highlighted a synergistic inhibition of mitochondrial energy metabolism (pyruvate dehydrogenase complex, tricarboxylic acid cycle, respiratory chain, mitochondrial salvage pathway of deoxyribonucleoside triphosphate (dNTP)) induced by propionyl-CoA, 2-methylcitrate and MMA as the key pathomechanism of inherited disorders of propionate metabolism. Intracerebral accumulation of toxic metabolites ('trapping' hypothesis') is considered a biochemical risk factor for neurodegeneration. Secondary effects of mitochondrial dysfunction, such as oxidative stress and impaired mtDNA homeostasis, contribute to pathogenesis of these disorders. The underlying pathomechanisms of chronic renal insufficiency in methylmalonic acidurias are not yet understood. We hypothesize that renal and cerebral pathomechanisms share some similarities, such as an involvement of dicarboxylic acid transport. This review aims to give a comprehensive overview on recent pathomechanistic concepts for methylmalonic acidurias.

Validation of an HPLC method for quantitation of MDMA in tablets.

An isocratic reversed-phase high-performance liquid chromatography (HPLC) method is developed and validated for the quantitation of 3,4-methylenedioxymethamphetamine (MDMA) in tablets. The chromatographic separation is achieved with potassium phosphate buffer (pH 3.2)-acetonitrile (9:1, v/v) as mobile phase, a Chromspher B column, and UV detection at 210 nm. The calibration curve is linear from 1.4 to 111 microg/mL. The percent relative standard deviation for intra- and interday precision studies is 2.7% each. The measurement uncertainty is estimated to 9%. The method is specific and successfully used for routine quantitation of MDMA in tablets.

Microsolvation of F- in water.

Microsolvation of F- in water is studied by ionization and double ionization spectra of (H2O)1-3F- calculated by ab initio methods. It is shown that the presence of the fluorine electrons introduces many-body properties in the spectra which cannot be reproduced by the presence of a negative point charge. The increase of the solvation shell increases the complexity in particular of the double ionization spectra. Ionization and double ionization energies slowly increase with continued solvation, and many-body effects in the inner valence spectra become more prominent.

Fatal overdose of the herbicide bentazone.

A 59-year-old woman who intentionally ingested 100-200 ml Basagran was taken to the hospital with a cardiac arrest 2 days after she had consumed the herbicide. During this period she suffered vomiting, urination and diarrhoea and she was drowsy with a muddled speech. Biological samples obtained at the autopsy were analysed and presence of bentazone, alcohol and an active metabolite of citalopram were detected. Blood concentrations of bentazone, alcohol and desmethyl-citalopram were 625 mg/kg, 0.62 g/l and 0.03 mg/kg, respectively.

[Substances other than alcohol found in the blood among Danish road-users during a week in 1996]. / Andre stoffer end alkohol i blodet hos danske trafikanter. En undersøgelse af trafiksager fra en uge i 1996.

INTRODUCTION: This investigation is the Danish contribution to a Nordic comparative study of other drugs than alcohol found in road users. MATERIAL AND METHODS: 255 blood samples received from the police for alcohol and/or drug determination during one week in 1996 were investigated for drugs. RESULTS: 56 (22%) of the 255 blood samples were positive for other drugs than alcohol. Cannabis was detected in 11%, benzodiazepines, mainly diazepam and flunitrazepam, in 9% and amphetamine in 5% of the blood samples. DISCUSSION: The police suspected other drugs than alcohol in 3% of the 255 blood samples, but drugs were detected in 22%. This investigation showed that the frequency of other drugs than alcohol was similar in Denmark and Norway for the blood samples with an alcohol concentration above the statutory limit. This contrasts with the fact that the number of blood samples from road users investigated for substances other than alcohol is about 200/year in Denmark and 4000/year in Norway.

[Fatal intoxications among drug addicts in Denmark in 1997]. / Forgiftningsdødsfald blandt narkomaner i Danmark i 1997.

INTRODUCTION: The purpose of this study was to investigate fatal poisonings among drug addicts in 1997 and to compare the results to similar investigations from 1985 and 1991. MATERIAL AND METHODS: All fatal intoxications among drug addicts in Denmark in 1997, investigated at the three Institutes of Forensic Medicine in Denmark. RESULTS: The number of fatal intoxications increased by 32% from 1991 to 1997, mainly outside the metropolitan area, The average age increased from 32 to 36 years. The proportion of heroin/morphine intoxications increased from 57% to 71%. The most commonly used drugs were as in 1991 heroin/morphine, diazepam and methadone. The frequency of cocaine increased from one positive case in 1991 to 14% positive cases in 1997. DISCUSSION: This study showed an increasing number of fatal intoxications and changes in drug abuse pattern and place of death since 1991.