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Background: We previously demonstrated that APR-246 (eprenetapopt) could be an efficient treatment option against neuroblastoma (NB), the most common pediatric extracranial solid tumor. APR-246's mechanism of action is not completely understood and can differ between cell types. Here we investigate the involvement of well-known oncogenic pathways in NB's response to APR-246., Methods: A proteome profiler kinase assays and western blot analysis were used to identify the molecular pathways involved in the responses to APR-246. Bulk ATP levels were used to determine the viability of cells and the IC 50 for APR-246. Cystine-FITC was used to measure the cellular uptake of cysteine. PmRNA5 was used to activate ERK1/2 and pshRNA1 was used to silence HSP27. An IMR-32 xenograft zebrafish embryo model was used to assess APR-246 and sulfasalazine efficacy in vivo ., Results: After APR-246 treatment, the most deregulated signaling protein identified was ERK1/2, an end-point kinase of the RAS-MAPK pathway. Induction of phospho-ERK1/2 resulted in increased glutathione (GSH) levels, increased cystine uptake, and increased resistance of NB cells to APR-246. Using ERK1/2 inhibitors in combination with APR-246, we were able to categorize cells into synergistic and antagonistic groups. After co-treatment, these two groups differ by their levels of SLC7A11 and Hsp27 phosphorylation, cystine uptake, and BIM expression. Using erastin and sulfasalazine, both inhibitors of SLC7A11 and activators of ferroptosis, we were able to reverse the antagonistic effects of ERK1/2 inhibitors and demonstrate a strong synergistic action in vitro and in vivo in zebrafish models., Conclusions: These results demonstrated a pivotal role of the RAS-MAPK pathway in the NB cellular response to APR-246 via the modulation of intracellular concentrations of GSH and the transport of cystine through SLC7A11, phosphorylation of Hsp27, and programmed cell death. Combining APR-246 with RAS-MAPK pathway inhibitors can, in some cases, lead to antagonistic action, which can be reversed by combining APR-246 with the clinically approved drug sulfasalazine., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Mlakar, Oehme, Lesne, Najafi, Ansari and Gumy-Pause.)

Cloning is a key molecular biology procedure for obtaining a genetically homogenous population of organisms or cell lines. It requires the expansion of new cell populations starting from single genetically modified cells. Despite the technical progress, cloning of many cell lines remains difficult. Cloning often fails either due to the strenuous conditions associated with manipulating cells or because many cells don't tolerate a single-cell state. Here we describe a new cloning method utilizing low adhesion microcavity plates. This new technique, named microcavity-assisted cloning (MAC) was developed to clone difficult-to-clone HepG2 cells. The clones were produced following CRISPR/Cas9 knockout of the GSTA1 gene by a random distribution of 200, 400, and 800 cells into 550 microcavities of a 24-well low adhesion plate originally designed for the culture of spheroids. The knockout of GSTA1 was verified at the protein level using Western blotting. The advantages of the MAC method are its low cost, ease of the procedure, and the possibility of scaling up the throughput and automatization., (© 2024. The Author(s).)

Crop type observation is crucial for various environmental and agricultural remote sensing applications including land use and land cover mapping, crop growth monitoring, crop modelling, yield forecasting, disease surveillance, and climate modelling. Quality-controlled georeferenced crop type information is essential for calibrating and validating machine learning algorithms. However, publicly available field data is scarce, particularly in the highly dynamic smallholder farming systems of sub-Saharan Africa. For the 2020/21 main cropping season ( Meher ), the Ethiopian Crop Type 2020 ( EthCT2020 ) dataset compiled from multiple sources provides 2,793 harmonized, quality-controlled, and georeferenced in-situ samples on annual crop types (7 crop groups; 22 crop classes) at smallholder field level across the complex and highly fragmented agricultural landscape of Ethiopia. The focus was on rainfed, wheat-based farming systems. A nationwide ground data collection campaign (GDCC; Source 1 ) was designed using a stratification approach based on wheat crop calendar information, and 1,263 in-situ data samples were collected in selected sampling regions. This in-situ data pool was enriched with 1,530 wheat samples extracted from a) the Wheat Rust Toolbox (WRTB; Source 2 ; 734 samples), a database for wheat disease surveillance data [1] and b) an inhouse farm household survey database (FHSD; Source 3 ; 796 samples). Obtained field data was labelled according to the Joint Experiment for Crop Assessment and Monitoring (JECAM) guidelines for cropland and crop type definition and field data collection [2] and the FAO Indicative Crop Classification [3]. The EthCT2020 dataset underwent extensive processing including data harmonization, mixed pixel assessment through visual interpretation using 5 m Planet satellite image composites, and quality-control using Sentinel-2 NDVI homogeneity analysis. The EthCT2020 dataset is unique in terms of crop diversity, pixel purity, and spatial accuracy while targeting a countrywide distribution. It is representative of Ethiopia's complex and highly fragmented agricultural landscape and can be useful for developing new machine learning algorithms for land use land cover mapping, crop type mapping, agricultural monitoring, and yield forecasting in smallholder cropping systems. The dataset can also serve as a baseline input parameter for crop models, climate models, and crop disease and pest forecasting systems., (© 2024 The Author(s).)

GSTA1 encodes a member of a family of enzymes that function to add glutathione to target electrophilic compounds, including carcinogens, therapeutic drugs, environmental toxins, and products of oxidative stress. GSTA1 has several functional SNPs within its promoter region that are responsible for a change in its expression by altering promoter function. This study aims to investigate distributions of GSTA1 promoter haplotypes across different human populations and to assess their impact on the expression of GSTA1. PHASE 2.1.1 was used to infer haplotypes and diplotypes of six GSTA1 promoter SNPs on 2501 individuals from 26 populations classified by the 1000 Genomes Project into five super-populations that included Africa (N = 660), America (N = 347), East Asia (N = 504), Europe (N = 502), and South Asia (N = 488). We used pairwise FST analysis to compare sub-populations and luciferase reporter assay (LRA) to evaluate the impact of each SNP on activation of transcription and interaction with other SNPs. The distributions of GSTA1 promoter haplotypes and diplotypes were significantly different among the different human populations. Three new promoter haplotypes were found in the African super-population. LRA demonstrated that SNPs at -52 and -69 has the most impact on GSTA1 expression, however other SNPs have a significant impact on transcriptional activity. Based on LRA, a new model of cis-elements interaction is presented. Due to the significant differences in GSTA1 diplotype population frequencies, future pharmacogenomics or disease-related studies would benefit from the inclusion of the complete GSTA1 promoter haplotype based on the newly proposed metabolic grouping derived from the LRA results.

Sinusoidal obstruction syndrome (SOS) is a well-recognized and potentially life-threatening complication of hematopoietic stem cell transplantation (HSCT). SOS arises from endothelial cell damage and hepatocellular injury mostly due to the transplantation conditioning regimens but also to other patient, disease, and treatment-related factors. Understanding risk factors associated with the development of SOS is critical for early initiation of treatment or prophylaxis. The knowledge about genetic contribution is limited; few studies investigated so far selected a set of genes. To get more comprehensive insight in the genetic component, we performed an exome-wide association study using genetic variants derived from whole-exome sequencing. The analyses were performed in a discovery cohort composed of 87 pediatric patients undergoing HSCT following a busulfan-containing conditioning regimen. Eight lead single-nucleotide polymorphisms (SNPs) were identified after correction for multiple testing and subsequently analyzed in a validation cohort (n = 182). Three SNPs were successfully replicated, including rs17146905 (P = .001), rs16931326 (P = .04), and rs2289971 (P = .03), located respectively in the UGT2B10, BHLHE22, and KIAA1715 genes. UGT2B10 and KIAA1715 were retained in a multivariable model while controlling for nongenetic covariates and previously identified risk variants in the GSTA1 promoter. The modulation of associations by conditioning regimens was noted; KIAA1715 was dependent on the intensity of the conditioning regimen, whereas the effect of UGT2B10 was equally applicable to all of them. Combined effect of associated loci was also observed (P = .00006) with a genotype-related SOS risk of 9.8. To our knowledge, this is the first study addressing the genetic component of SOS at an exome-wide level and identifying novel genetic variations conferring a higher risk of SOS, which might be useful for personalized prevention and treatment strategies., (Copyright © 2019 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Background: Neuroblastoma is the most common extracranial solid tumor in children. This cancer has a low frequency of TP53 mutations and its downstream pathway is usually intact. This study assessed the efficacy of the p53 activator, PRIMA-1 MET , in inducing neuroblastoma cell death., Methods: CellTiter 2.0 was used to study susceptibility and specificity of NB cell lines to PRIMA-1 MET . Real-time PCR and western blot were used to assess the most common p53 transactivation targets. Induction of p53 and Noxa, and inhibition of Cas3/7, were used to assess impact on cell death after PRIMA-1 MET treatment. Flow cytometry was used to analyze cell cycle phase and induction of apoptosis, reactive oxygen species, and the collapse of mitochondrial membrane potential., Results: Neuroblastoma cell lines were at least four times more susceptible to PRIMA-1 MET than were primary fibroblasts and keratinocyte cell lines. PRIMA-1 MET induced cell death rapidly and in all cell cycle phases. Although PRIMA-1 MET activated p53 transactivation activity, p53's role is likely limited because its main targets remained unaffected, whereas pan-caspase inhibitor demonstrated no ability to prevent cell death. PRIMA-1 MET induced oxidative stress and modulated the methionine/cysteine/glutathione axis. Variations of MYCN and p53 modulated intracellular levels of GSH and resulted in increased/decreased sensitivity of PRIMA-1 MET . PRIMA-1 MET inhibited thioredoxin reductase, but the effect of PRIMA-1 MET was not altered by thioredoxin inhibition., Conclusions: PRIMA-1 MET could be a promising new agent to treat neuroblastoma because it demonstrated good anti-tumor action. Although p53 is involved in PRIMA-1 MET -mediated cell death, our results suggest that direct interaction with p53 has a limited role in neuroblastoma but rather acts through modulation of GSH levels.

Several studies have reported an association between the farnesoid X receptor alpha (FXRα) and estrogenic signaling pathways. Fxrα could thus be involved in the reprotoxic effects of endocrine disruptors such as bisphenol-A (BPA). To test this hypothesis, mice were exposed to BPA and/or stigmasterol (S), an FXRα antagonist. Following the exposure to both molecules, wild-type animals showed impaired fertility and lower sperm cell production associated with the alteration of the establishment and maintenance of the undifferentiated germ cell pool. The crosstalk between BPA and FXRα is further supported by the lower impact of BPA in mice genetically ablated for Fxrα and the fact that BPA counteracted the effects of FXRα agonists. These effects might result from the downregulation of Fxrα expression following BPA exposure. BPA and S act additively in human testis. Our data demonstrate that FXRα activity modulates the impact of BPA on male gonads and on undifferentiated germ cell population., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Busulfan (BU) dose adjustment following therapeutic drug monitoring contributes to better outcome of hematopoietic stem cell transplantation (HSCT). Further improvement could be achieved through genotype-guided BU dose adjustments. To investigate this aspect, polymorphism within glutathione S transferase genes were assessed. Particularly, promoter haplotypes of the glutathione S transferase A1 ( GSTA1 ) were evaluated in vitro, with reporter gene assays and clinically, in a pediatric multi-center study (N =138) through association with BU pharmacokinetics (PK) and clinical outcomes. Promoter activity significantly differed between the GSTA1 haplotypes (p<0.001) supporting their importance in capturing PK variability. Four GSTA1 diplotype groups that significantly correlated with clearance (p=0.009) were distinguished. Diplotypes underlying fast and slow metabolizing capacity showed higher and lower BU clearance (ml/min/kg), respectively. GSTA1 diplotypes with slow metabolizing capacity were associated with higher incidence of sinusoidal obstruction syndrome, acute graft versus host disease and combined treatment-related toxicity (p<0.0005). Among other GST genes investigated, GSTP1 313GG correlated with acute graft versus host disease grade 1-4 (p=0.01) and GSTM1 non-null genotype was associated with hemorrhagic cystitis (p=0.003). This study further strengthens the hypothesis that GST diplotypes/genotypes could be incorporated into already existing population pharmacokinetic models for improving first BU dose prediction and HSCT outcomes. (N o Clinicaltrials.gov identifier: NCT01257854. Registered 8 December 2010, retrospectively registered)., Competing Interests: CONFLICTS OF INTEREST H.B has acted as a consultant for Jazz Pharmaceuticals and obtained an education grant from them. H.B also acted as a consultant for Seattle Genetics. The authors declare that they have no other financial relationship(s) to disclose.

Neuroblastoma, a childhood cancer with highly heterogeneous biology and clinical behavior, is characterized by genomic aberrations including amplification of MYCN. Hemizygous deletion of chromosome 11q is a well-established, independent marker of poor prognosis. While 11q22-q23 is the most frequently deleted region, the neuroblastoma tumor suppressor in this region remains to be identified. Chromosome bands 11q22-q23 contain ATM, a cell cycle checkpoint kinase and tumor suppressor playing a pivotal role in the DNA damage response. Here, we report that haploinsufficiency of ATM in neuroblastoma correlates with lower ATM expression, event-free survival, and overall survival. ATM loss occurs in high stage neuroblastoma without MYCN amplification. In SK-N-SH, CLB-Ga and GI-ME-N human neuroblastoma cells, stable ATM silencing promotes neuroblastoma progression in soft agar assays, and in subcutaneous xenografts in nude mice. This effect is dependent on the extent of ATM silencing and does not appear to involve MYCN. Our findings identify ATM as a potential haploinsufficient neuroblastoma tumor suppressor, whose inactivation mirrors the increased aggressiveness associated with 11q deletion in neuroblastoma.

Aluminium salts used as antiperspirants have been incriminated as contributing to breast cancer incidence in Western societies. To date, very little or no epidemiological or experimental data confirm or infirm this hypothesis. We report here that in MCF-10A human mammary epithelial cells, a well-established normal human mammary epithelial cell model, long-term exposure to aluminium chloride (AlCl(3) ) concentrations of 10-300 µ m, i.e. up to 100 000-fold lower than those found in antiperspirants, and in the range of those recently measured in the human breast, results in loss of contact inhibition and anchorage-independent growth. These effects were preceded by an increase of DNA synthesis, DNA double strand breaks (DSBs), and senescence in proliferating cultures. AlCl(3) also induced DSBs and senescence in proliferating primary human mammary epithelial cells. In contrast, it had no similar effects on human keratinocytes or fibroblasts, and was not detectably mutagenic in bacteria. MCF-10A cells morphologically transformed by long-term exposure to AlCl(3) display strong upregulation of the p53/p21(Waf1) pathway, a key mediator of growth arrest and senescence. These results suggest that aluminium is not generically mutagenic, but similar to an activated oncogene, it induces proliferation stress, DSBs and senescence in normal mammary epithelial cells; and that long-term exposure to AlCl(3) generates and selects for cells able to bypass p53/p21(Waf1) -mediated cellular senescence. Our observations do not formally identify aluminium as a breast carcinogen, but challenge the safety ascribed to its widespread use in underarm cosmetics., (Copyright © 2012 John Wiley & Sons, Ltd.)

Carriers of mutations in the cell cycle checkpoint protein kinase ataxia telangiectasia mutated (ATM), which represent 1-2% of the general population, have an increased risk of breast cancer. However, experimental evidence that ATM deficiency contributes to human breast carcinogenesis is lacking. We report here that in MCF-10A and MCF-12A cells, which are well established normal human mammary gland epithelial cell models, partial or almost complete stable ATM silencing or pharmacological inhibition resulted in cellular transformation, genomic instability, and formation of dysplastic lesions in NOD/SCID mice. These effects did not require the activity of exogenous DNA-damaging agents and were preceded by an unsuspected and striking increase in cell proliferation also observed in primary human mammary gland epithelial cells. Increased proliferation correlated with a dramatic, transient, and proteasome-dependent reduction of p21(WAF1/CIP1) and p27(KIP1) protein levels, whereas little or no effect was observed on p21(WAF1/CIP1) or p27(KIP1) mRNAs. p21(WAF1/CIP1) silencing also increased MCF-10A cell proliferation, thus identifying p21(WAF1/CIP1) down-regulation as a mediator of the proliferative effect of ATM inhibition. Our findings provide the first experimental evidence that ATM is a human breast tumor suppressor. In addition, they mirror the sensitivity of ATM tumor suppressor function and unveil a new mechanism by which ATM might prevent human breast tumorigenesis, namely a direct inhibitory effect on the basal proliferation of normal mammary epithelial cells.