CKLF instigates a "cold" microenvironment to promote MYCN-mediated tumor aggressiveness.

Solid tumors, especially those with aberrant MYCN activation, often harbor an immunosuppressive microenvironment to fuel malignant growth and trigger treatment resistance. Despite this knowledge, there are no effective strategies to tackle this problem. We found that chemokine-like factor (CKLF) is highly expressed by various solid tumor cells and transcriptionally up-regulated by MYCN. Using the MYCN-driven high-risk neuroblastoma as a model system, we demonstrated that as early as the premalignant stage, tumor cells secrete CKLF to attract CCR4-expressing CD4+ cells, inducing immunosuppression and tumor aggression. Genetic depletion of CD4+ T regulatory cells abolishes the immunorestrictive and protumorigenic effects of CKLF. Our work supports that disrupting CKLF-mediated cross-talk between tumor and CD4+ suppressor cells represents a promising immunotherapeutic approach to battling MYCN-driven tumors.

A coupled flow and transport model for simulation of multi-species reactive transport in unconfined aquifer using meshless local Petrov Galerkin (MLPG) method.

An understanding of natural degradation of multiple reactive contaminants in the aquifers is essential before designing the monitoring or remediation programs for polluted aquifers. Since such reactive contaminants are ubiquitous, a number of research works has been performed in the past three decades for the modelling of multi-species reactive transport (MSRT) phenomenon. The widely used finite difference method (FDM) and finite element method (FEM)-based models suffer a drawback of relying on a grid/mesh, which makes the solution unstable. Addressing such difficulties, the latest research on the MSRT models is directed towards the meshless methods. In this study, the meshless local Petrov Galerkin (MLPG) method-based multi-species reactive transport model (MLPG-MSRT) is presented, with an objective to create a robust simulation tool for the prediction of fate of multiple contaminants of the first-order reaction network. The developed model is validated for reversible as well as irreversible reaction networks with the available analytical solutions. Also, the MLPG model for unconfined aquifer flow (UF) is developed, validated, and coupled with the MLPG-MSRT model. The MLPG-UF-MSRT model results are further compared with the established FDM-based MODFLOW-RT3D model solutions for a rectangular and a real field type study. The results showed that the proposed model can simulate MSRT as accurately as the FDM-based models with an additional advantage of simplicity and stability, and thus, is more efficient for complex field problems.

Epigenetic modulation of neuroblastoma enhances T cell and NK cell immunogenicity by inducing a tumor-cell lineage switch.

BACKGROUND: Immunotherapy in high-risk neuroblastoma (HR-NBL) does not live up to its full potential due to inadequate (adaptive) immune engagement caused by the extensive immunomodulatory capacity of HR-NBL. We aimed to tackle one of the most notable immunomodulatory processes in neuroblastoma (NBL), absence of major histocompatibility complex class I (MHC-I) surface expression, a process greatly limiting cytotoxic T cell engagement. We and others have previously shown that MHC-I expression can be induced by cytokine-driven immune modulation. Here, we aimed to identify tolerable pharmacological repurposing strategies to upregulate MHC-I expression and therewith enhance T cell immunogenicity in NBL. METHODS: Drug repurposing libraries were screened to identify compounds enhancing MHC-I surface expression in NBL cells using high-throughput flow cytometry analyses optimized for adherent cells. The effect of positive hits was confirmed in a panel of NBL cell lines and patient-derived organoids. Compound-treated NBL cell lines and organoids were cocultured with preferentially expressed antigen of melanoma (PRAME)-reactive tumor-specific T cells and healthy-donor natural killer (NK) cells to determine the in vitro effect on T cell and NK cell cytotoxicity. Additional immunomodulatory effects of histone deacetylase inhibitors (HDACi) were identified by transcriptome and translatome analysis of treated organoids. RESULTS: Drug library screening revealed MHC-I upregulation by inhibitor of apoptosis inhibitor (IAPi)- and HDACi drug classes. The effect of IAPi was limited due to repression of nuclear factor kappa B (NFκB) pathway activity in NBL, while the MHC-I-modulating effect of HDACi was widely translatable to a panel of NBL cell lines and patient-derived organoids. Pretreatment of NBL cells with the HDACi entinostat enhanced the cytotoxic capacity of tumor-specific T cells against NBL in vitro, which coincided with increased expression of additional players regulating T cell cytotoxicity (eg, TAP1/2 and immunoproteasome subunits). Moreover, MICA and MICB, important in NK cell cytotoxicity, were also increased by entinostat exposure. Intriguingly, this increase in immunogenicity was accompanied by a shift toward a more mesenchymal NBL cell lineage. CONCLUSIONS: This study indicates the potential of combining (immuno)therapy with HDACi to enhance both T cell-driven and NKcell-driven immune responses in patients with HR-NBL.

Simulation of reactive transport in porous media using Meshless Local Petrov Galerkin (MLPG) and combination of Meshless Weak and Strong (MWS) form methods.

In this paper, two meshless methods, namely, a weak form Meshless Local Petrov Galerkin (MLPG) method, and Meshless Weak Strong (MWS) form method, obtained by combining MLPG with a strong form Radial Point Collocation Method (RPCM), are presented for simulation of advection-dispersion-reaction phenomena of the contaminants in the porous media. The first-order decay and sorption reactions are considered in this study. The Crank Nicolson scheme is applied for the time discretization. The weak form MLPG is a truly meshless and robust numerical technique, that can be applied to complex aquifer systems with derivative boundaries. However, in this method, the computational time is increased due to the integration, which is not essential for simple problems. Thus, the MLPG method is further coupled with a strong form RPCM with an aim of decreasing the background integration, by modelling only the nodes around the derivative boundaries using MLPG method and the other nodes by a direct RPCM which do not require integration. The proposed MWS model automatically converts into a complete RPCM model if there are no derivative boundaries. Thus, this model being both accurate and computationally efficient is suitable for simple and moderately complex aquifer systems and MLPG is the most stable and reliable method for modelling the most complex aquifer problems. Both the developed models are tested with available analytical solutions and applied for hypothetical case studies. The results prove the efficiency of the models and the applicability of each model is described in detail.

Mesenchymal and adrenergic cell lineage states in neuroblastoma possess distinct immunogenic phenotypes.

Apart from the anti-GD2 antibody, immunotherapy for neuroblastoma has had limited success due to immune evasion mechanisms, coupled with an incomplete understanding of predictors of response. Here, from bulk and single-cell transcriptomic analyses, we identify a subset of neuroblastomas enriched for transcripts associated with immune activation and inhibition and show that these are predominantly characterized by gene expression signatures of the mesenchymal lineage state. By contrast, tumors expressing adrenergic lineage signatures are less immunogenic. The inherent presence or induction of the mesenchymal state through transcriptional reprogramming or therapy resistance is accompanied by innate and adaptive immune gene activation through epigenetic remodeling. Mesenchymal lineage cells promote T cell infiltration by secreting inflammatory cytokines, are efficiently targeted by cytotoxic T and natural killer cells and respond to immune checkpoint blockade. Together, we demonstrate that distinct immunogenic phenotypes define the divergent lineage states of neuroblastoma and highlight the immunogenic potential of the mesenchymal lineage.

Synergistic Anti-Tumor Effect of Combining Selective CDK7 and BRD4 Inhibition in Neuroblastoma.

PURPOSE: Cyclin-dependent kinases (CDKs) that have critical roles in RNA polymerase II (Pol II)-mediated gene transcription are emerging as therapeutic targets in cancer. We have previously shown that THZ1, a covalent inhibitor of CDKs 7/12/13, leads to cytotoxicity in MYCN-amplified neuroblastoma through the downregulation of super-enhancer-associated transcriptional upregulation. Here we determined the effects of YKL-5-124, a novel covalent inhibitor with greater selectivity for CDK7 in neuroblastoma cells. EXPERIMENTAL DESIGN: We tested YKL-5-124 in MYCN-amplified and nonamplified neuroblastoma cells individually and in combination with other inhibitors in cell line and animal models. Cell viability, target validation, effects on cell cycle and transcription were analyzed. RESULTS: CDK7 inhibition with YKL-5-124 did not lead to significant cell death, but resulted in aberrant cell cycle progression especially in MYCN-amplified cells. Unlike THZ1, YKL-5-124 had minimal effects on Pol II C-terminal domain phosphorylation, but significantly inhibited that of the CDK1 and CDK2 cell cycle kinases. Combining YKL-5-124 with the BRD4 inhibitor JQ1 resulted in synergistic cytotoxicity. A distinct MYCN-gene expression signature associated with resistance to BRD4 inhibition was suppressed with the combination. The synergy between YKL-5-124 and JQ1 translated into significant tumor regression in cell line and patient-derived xenograft mouse models of neuroblastoma. CONCLUSIONS: The combination of CDK7 and BRD4 inhibition provides a therapeutic option for neuroblastoma and suggests that the addition of YKL-5-124 could improve the therapeutic efficacy of JQ1 and delay resistance to BRD4 inhibition.

Short test of mental status in the detection of mild cognitive impairment in India.

CONTEXT: Mild cognitive impairment (MCI) is an under-diagnosed health problem in the community. Cognitive screening tools are widely used for MCI detection, but many of them lack sensitivity and specificity in MCI detection. On the basis of literature review, Short test of mental status (STMS) was selected for the present purpose. AIMS: The present study purports to standardize STMS for using as a cognitive screening tool in MCI detection in the community-living elderly people in Kolkata. MATERIALS AND METHODS: Data were collected from 102 community-living elderly people from the city of Kolkata using the purposive method of sampling. MCI was diagnosed using the Peterson's criteria of MCI as the gold standard. A semi-structured demographic proforma, clock-drawing test (CDT), and the Groningen Activity Restriction Scale (GARS) were used for the purpose. Finally, STMS was administered. STATISTICAL ANALYSIS: Statistical computation was done using the SPSS 21. Descriptive statistics, receiver operating curve analysis, and binary logistic regression were used for statistical analysis of the data. RESULTS AND CONCLUSION: STMS emerged as a sensitive and specific cognitive screening tool in the detection of MCI in the current cultural setting. It was also found to be more suited for the purpose than CDT. A score of 34.5 with a sensitivity of 97.5 and a specificity of 90.3% were selected for the optimum cutoff score for the detection of MCI in the present population. With a unit increase in STMS score, the odds of getting diagnosed with MCI were found to be reduced by 48.5%.

Variation of gene expression in plants is influenced by gene architecture and structural properties of promoters.

In higher eukaryotes, gene architecture and structural properties of promoters have emerged as significant factors influencing variation in number of transcripts (expression level) and specificity of gene expression in a tissue (expression breadth), which eventually shape the phenotype. In this study, transcriptome data of different tissue types at various developmental stages of A. thaliana, O. sativa, S. bicolor and Z. mays have been used to understand the relationship between properties of gene components and its expression. Our findings indicate that in plants, among all gene architecture and structural properties of promoters, compactness of genes in terms of intron content is significantly linked to gene expression level and breadth, whereas in human an exactly opposite scenario is seen. In plants, for the first time we have carried out a quantitative estimation of effect of a particular trait on expression level and breadth, by using multiple regression analysis and it confirms that intron content of primary transcript (as %) is a powerful determinant of expression breadth. Similarly, further regression analysis revealed that among structural properties of the promoters, stability is negatively linked to expression breadth, while DNase1 sensitivity strongly governs gene expression breadth in monocots and gene expression level in dicots. In addition, promoter regions of tissue specific genes are found to be enriched with TATA box and Y-patch motifs. Finally, multi copy orthologous genes in plants are found to be longer, highly regulated and tissue specific.

Genome-Wide Targets Regulated by the OsMADS1 Transcription Factor Reveals Its DNA Recognition Properties.

OsMADS1 controls rice (Oryza sativa) floral fate and organ development. Yet, its genome-wide targets and the mechanisms underlying its role as a transcription regulator controlling developmental gene expression are unknown. We identify 3112 gene-associated OsMADS1-bound sites in the floret genome. These occur in the vicinity of transcription start sites, within gene bodies, and in intergenic regions. Majority of the bound DNA contained CArG motif variants or, in several cases, only A-tracts. Sequences flanking the binding peak had a higher AT nucleotide content, implying that broader DNA structural features may define in planta binding. Sequences for binding by other transcription factor families like MYC, AP2/ERF, bZIP, etc. are enriched in OsMADS1-bound DNAs. Target genes implicated in transcription, chromatin remodeling, cellular processes, and hormone metabolism were enriched. Combining expression data from OsMADS1 knockdown florets with these DNA binding data, a snapshot of a gene regulatory network was deduced where targets, such as AP2/ERF and bHLH transcription factors and chromatin remodelers form nodes. We show that the expression status of these nodal factors can be altered by inducing the OsMADS1-GR fusion protein and present a model for a regulatory cascade where the direct targets of OsMADS1, OsbHLH108/SPT, OsERF034, and OsHSF24, in turn control genes such as OsMADS32 and OsYABBY5 This cascade, with other similar relationships, cumulatively contributes to floral organ development. Overall, OsMADS1 binds to several regulatory genes and, probably in combination with other factors, controls a gene regulatory network that ensures rice floret development.