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Climate change, hydro-power dams and socio-economic development in the upper Mekong River, have caused changes in surface water resources and agricultural transformation in the Vietnamese Mekong Delta (VMD). It is unpredictable and influences decision-making processes on water resource management. This study aims at applying the DAPP (Dynamic Adaptive Policy Pathways) approach to develop solution roadmaps supporting rice production in the Mekong Delta projection to 2030 under the context of uncertain changes on surface water resources. Data were collected from local officers, farmers, experts at Can Tho University, Technical University Delft, and Utrecht University using semi-structured interview techniques including workshops, group discussions, and in-depth interviews. The consolidated solutions from stakeholder’s opinions were evaluated at different socio-economic and environmental dimensions, specially focused on the time of tipping point of each solution. The study results show that two solution roadmaps were developed as sample scenarios to promote rice production in the upper area of the VMD. The determination of the solutions roadmaps based on the agreement of the stakeholders, which is a new feature of this approach in linking many stakeholders for the general development of the region. The DAPP approach could be suitable to support decision-making on surface water resources management in the context of uncertain changes. Therefore, further studies can apply this approach to other regions in the VMD which will support to reinforce and demonstrate the applicability of the DAPP approach on supporting decision-making in the face of increasingly uncertain changes in the VMD.

DNA methyltransferase inhibitors (DNMTI) like 5-Azacytidine (5-Aza) are the only disease-modifying drugs approved for the treatment of higher-risk myelodysplastic syndromes (MDS), however less than 50% of patients respond, and there are no predictors of response with clinical utility. Somatic mutations in the DNA methylation regulating gene tet-methylcytosine dioxygenase 2 (TET2) are associated with response to DNMTIs, however the mechanisms responsible for this association remain unknown. Using bisulfite padlock probes, mRNA sequencing, and hydroxymethylcytosine pull-down sequencing at several time points throughout 5-Aza treatment, we show that TET2 loss particularly influences DNA methylation (5mC) and hydroxymethylation (5hmC) patterns at erythroid gene enhancers and is associated with downregulation of erythroid gene expression in the human erythroleukemia cell line TF-1. 5-Aza disproportionately induces expression of these down-regulated genes in TET2KO cells and this effect is related to dynamic 5mC changes at erythroid gene enhancers after 5-Aza exposure. We identified differences in remethylation kinetics after 5-Aza exposure for several types of genomic regulatory elements, with distal enhancers exhibiting longer-lasting 5mC changes than other regions. This work highlights the role of 5mC and 5hmC dynamics at distal enhancers in regulating the expression of differentiation-associated gene signatures, and sheds light on how 5-Aza may be more effective in patients harboring TET2 mutations.Implications:TET2 loss in erythroleukemia cells induces hypermethylation and impaired expression of erythroid differentiation genes which can be specifically counteracted by 5-Azacytidine, providing a potential mechanism for the increased efficacy of 5-Aza in TET2-mutant patients with MDS.Visual Overview:http://mcr.aacrjournals.org/content/molcanres/19/3/451/F1.large.jpg.

A) Expression of hemoglobin genes which increase in terminally differentiated erythroid cells. B) Expression of key erythrocyte marker genes. TPM=Transcripts per kilobase million; a gene expression measure for RNAseq that normalizes for gene-length and the sample sequencing depth.

A) Dose-response curve of wild-type TF1 cell viability for three different lengths of 5-Aza treatments (1 dose per day for a total length of treatment between 1 and 3 days). Points represent replicates per condition, shaded region corresponds to 95% confidence band estimated by 4 parameter logistic regression model (EC50 and slope as parameter and lower bounded at zero; modeled with R package "drc"). B) Dose-response of DNMT1 downregulation 24 hours after the indicated 5-Aza dose. C) Time course of DNMT1 downregulation following the indicated dosage schedules.

Cells were treated with 100uM L-AA or vehicle 24hrs before DNA isolation. A) 5hmC dot blot for L-AA treated or untreated Cells. B) Standard curve of 5hmC densitometric signal vs. pmol (calculated from 5hmC standard with known 5hmC amount). C) Quantification of dot blot signal for TET2-WT and KO cells +/- L-AA.

Line plots in grid: each colored line represents a single CpG locus over time after 5-Aza treatment. Black line indicates mean value for that cluster group. Dotted black line indicates mean +/- 1 standard deviation. From left to right: clustering solutions with K=2 to K=9 clusters (derived from cutting the hierarchical cluster tree at different levels); top to bottom: the patterns of methylation in each cluster for that cluster solution. Mean silhouette width plot shows the mean silhouette width of all clusters for each individual clustering solution. K=4 clusters produced the most well-defined clusters as indicated by the peak in silhouette width.

Here we describe a computational protocol for performing quality control analysis on shallow sequencing data obtained from 10X snRNA-seq experiments. The workflow starts with raw MiSeq run folders and uses cellranger to generate count matrices. The raw count matrices are analyzed and sequencing saturation plots are generated. The saturation plots are then compared against plots from a reference set of libraries with varying qualities (bad, fair, good, great), thus allowing for the determination of sequencing requirements as well as an assessment of the overall quality of each 10X snRNA experiment.

10X Genomics Single Cell 3' (v3) RNA sequencing is a microdroplet-based method that permits the effective capture and sequencing of the mRNA and pre-mRNA molecules from single nuclei [1]. RNA molecules are transcribed and processed within the nucleus before exporting to ER for translation into proteins. As such, nuclear RNA is a mixture of nascent transcripts, partially or fully processed mRNA, and various non-coding RNA molecules. The total RNA content within the nucleus is roughly 10% of the RNA content in a whole cell, but has been found to accurately represent whole cell expression values in adult human tissues [2,3] including the kidney [4]. 10x Genomics Single Cell ATAC sequencing is a microdroplet-based method that allows for the effective capture, sequencing and profiling of accessible chromatin in single nuclei. Chromatin accessibility is a major determinant of gene regulation, defining the transcriptional regulatory networks that determine cellular identity and function as well as additional biological processes (e.g., differentiation, proliferation, development and responses to the extracellular environment). The 10X Multiome ATAC + Gene Expression assay permits capture of both RNA expression and epigenomic profiles from the same nuclei for a deeper understanding of cell type or state gene regulation. Here we present a modified version of the published 10X protocol [5] that we have adapted for the processing of nuclei isolated from adult human organs or tissues (e.g. kidney). Nuclei can be readily isolated from frozen tissues with a combination of chemical and physical treatments that can effectively circumvent the non-uniform or incomplete dissociation of solid tissues into single cells, as well as RNA degradation or artefacts (such as stress response) during dissociation. This 10X Multiome ATAC + Gene Expression protocol permits the generation of a molecular atlas of a human organ or tissue with comprehensive cell types and minimal processing artifacts. References 1. Chromium Single Cell 3' Reagent Kits v3 User Guide (Rev A) CG000183, support.10xgenomics.com. 2. Lake et al. (2016). Science, doi:10.1126/science.aaf1204. 3. Lake et al. (2018). Nature Biotechnology, doi:10.1038/nbt.4038. 4. Lake et al. (2019). Nature Communications, doi:10.1038/s41467-019-10861-2. 5. Chromium Next GEM Single Cell Multiome ATAC + Gene Expression (CG000338 Rev D).

Neuroblastoma is a childhood cancer originating from embryonic neuronal progenitor cells and is the most common cancer diagnosed in infants. Although the majority of patients respond to initial chemotherapy, most high-risk patients suffer relapse due to therapy resistance. Here, we generated single-cell transcriptome and bulk whole-genome sequencing data of diagnosis and post-therapy samples from 23 patients diagnosed with high-risk neuroblastoma. We found two distinct adrenergic populations in the patient samples. Most tumor cells showed a mature sympathoblast phenotype whereas a small fraction was in an undifferentiated state with activation of protein translation, unfolded protein, and oxidative phosphorylation pathways. We found that therapy-resistant tumor cells in these two subpopulations had distinct characteristics. The more mature resistant subpopulation upregulated genes associated with epithelial-to-mesenchymal transition, including TWIST1, PLCB1 and CD276. The undifferentiated resistant subpopulation upregulated Ras signal transduction and TP53 pathway genes. Analysis of the immune microenvironment revealed that most tumor-associated macrophages became more immunosuppressive post-therapy via multiple newly gained signaling interactions including the complement signaling pathway. We discovered a limited infiltration of T lymphocytes in the tumor microenvironment, and chemotherapy induced an effector state with upregulated mTOR signaling and metabolism. Overall, our study revealed subpopulations of tumor cells in neuroblastoma that responded differently to induction chemotherapy. Our findings uncovered distinct molecular signatures of the resistant cells and their interactions with the immune microenvironment, paving the way for developing novel therapies for high-risk neuroblastoma. Citation Format: Yasin Uzun, Liron D. Grossmann, Chia-Hui Chen, Anusha Thadi, Chi-Yun Wu, Peng Gao, Dinh Diep, Lea Surrey, Daniel Martinez, Tasleema Patel, Qi Qiu, Sarah Johnson, Wenbao Yu, Shane Drabings, Changya Chen, Yuxuan Hu, Gregory Chen, Derek A. Oldridge, Kun Zhang, Hao Wu, Kathrin Bernt, Nancy Zhang, John M. Maris, Kai Tan. Longitudinal single-cell sequencing of high-risk neuroblastoma tumors reveals intrinsic and extrinsic mechanisms of therapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6051.

BACKGROUND High-risk neuroblastoma is a pediatric cancer arising from the developing sympathetic nervous system with a 50% relapse rate that is typically fatal. At least two subpopulations of neuroblastoma cells exist that can transdifferentiate, adrenergic and mesenchymal, the latter being more resistant to chemotherapy. Mechanisms of therapy resistance are largely unknown and the cells responsible for relapse have not been identified. METHODS We used single nucleus RNA and ATAC sequencing to identify and characterize the cells that survive chemotherapy, termed here “persister cells”, from a cohort of 20 matched diagnostic and post induction chemotherapyhigh-risk neuroblastoma patients and two patient derived xenograft (PDX) models from diagnostic tumors. Eight representative cell lines derived from neuroblastomas at diagnosis were treated with standard-of-care chemotherapy, and flow cytometry was used to sort for live cells. ML120B and CRISPR-CAS9 were used to modulate NF-kB signaling. An RNA-seq dataset of 153 high-risk neuroblastoma patients was used to determine differentially activated pathways between adrenergic and mesenchymal tumors. RESULTS Residual malignant cells in the post-chemotherapy tumor samples clustered into three main groups separated by the response to therapy. The most prevalent group of persister cells in responders (N=16/20) displayed low MYC(N) activity even in the presence of MYCN amplification. This group also demonstrated decreased expression of the adrenergic core regulatory circuit genes including PHOX2B, ISL1, HAND2, along with marked activation of TNF-alpha via NF-kB signaling. High NF-kB activity was found in a subpopulation of diagnostic cells in two chemo-refractory patients. We validated decreased expression of MYCN (2-fold decrease, p CONCLUSIONS NF-kB activation is a major mediator of de novo and acquired chemotherapy resistance in high-risk neuroblastoma. We postulate that concomitant silencing of this pathway could eliminate persister cells and prevent disease relapse. Citation Format: Liron D. Grossmann, Yasin Uzun, Jarrett Lindsay, Chia-Hui Chen, Catherine Wingrove, Peng Gao, Anusha Thadi, Quinlen Marshall, Nathan M. Kendsersky, Lea Surrey, Daniel Martinez, Emily Mycek, Colleen Casey, Kateryna Krytska, Matthew Tsang, Adam Wolpaw, David N. Groff, Erin Runbeck, Jayne McDevitt, Dinh Diep, Tasleema Patel, Kathrin M. Bernt, Chi Dang, Kun Zhang, Yael P. Mosse, Kai Tan, John M. Maris. NF-kB is a master regulator of resistance to therapy in high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 699.

Ninh Kieu District is located in the center of Can Tho City, established under the Government’s Decree No. 05/2004/ND-CP. This is a modern and rich district, with a history of formation and development associated with the Mekong Delta. According to statistics in 2019, Ninh Kieu District has an average population of 280,792 people, with a population density of 9,605 people/km2 and an area of 29.23 km2. In the current period, the urbanization process in Ninh Kieu District takes place at a fast and strong pace. This has made it impossible for the urban drainage system to keep up with this change, thereby creating great difficulties for the drainage management here. In order to deal with these enormous challenges, the managers in this area need to incorporate new technologies into drainage management. This paper presents the main result of building a spatial database to support drainage management for Ninh Kieu District. This is considered as a fundamental source of data for application of GIS in urban drainage research and management in Ninh Kieu District.