Oxidative treatment of micropollutants present in wastewater: A special emphasis on transformation products, their toxicity, detection, and field-scale investigations.

Micropollutants have become ubiquitous in aqueous environments due to the increased use of pharmaceuticals, personal care products, pesticides, and other compounds. In this review, the removal of micropollutants from aqueous matrices using various advanced oxidation processes (AOPs), such as photocatalysis, electrocatalysis, sulfate radical-based AOPs, ozonation, and Fenton-based processes has been comprehensively discussed. Most of the compounds were successfully degraded with an efficiency of more than 90%, resulting in the formation of transformation products (TPs). In this respect, degradation pathways with multiple mechanisms, including decarboxylation, hydroxylation, and halogenation, have been illustrated. Various techniques for the analysis of micropollutants and their TPs have been discussed. Additionally, the ecotoxicity posed by these TPs was determined using the toxicity estimation software tool (T.E.S.T.). Finally, the performance and cost-effectiveness of the AOPs at the pilot scale have been reviewed. The current review will help in understanding the treatment efficacy of different AOPs, degradation pathways, and ecotoxicity of TPs so formed.

Passenger Gene Coamplifications Create Collateral Therapeutic Vulnerabilities in Cancer.

DNA amplifications in cancer do not only harbor oncogenes. We sought to determine whether passenger coamplifications could create collateral therapeutic vulnerabilities. Through an analysis of >3,000 cancer genomes followed by the interrogation of CRISPR-Cas9 loss-of-function screens across >700 cancer cell lines, we determined that passenger coamplifications are accompanied by distinct dependency profiles. In a proof-of-principle study, we demonstrate that the coamplification of the bona fide passenger gene DEAD-Box Helicase 1 (DDX1) creates an increased dependency on the mTOR pathway. Interaction proteomics identified tricarboxylic acid (TCA) cycle components as previously unrecognized DDX1 interaction partners. Live-cell metabolomics highlighted that this interaction could impair TCA activity, which in turn resulted in enhanced mTORC1 activity. Consequently, genetic and pharmacologic disruption of mTORC1 resulted in pronounced cell death in vitro and in vivo. Thus, structurally linked coamplification of a passenger gene and an oncogene can result in collateral vulnerabilities. SIGNIFICANCE: We demonstrate that coamplification of passenger genes, which were largely neglected in cancer biology in the past, can create distinct cancer dependencies. Because passenger coamplifications are frequent in cancer, this principle has the potential to expand target discovery in oncology. This article is featured in Selected Articles from This Issue, p. 384.

Prediction of elevated groundwater fluoride across India using multi-model approach: insights on the influence of geologic and environmental factors.

Elevated fluoride in groundwater is a severe problem in India due to its extensive occurrence and detrimental health impacts on the large population that thrives on groundwater. Although fluoride is primarily a geogenic pollutant, existing model-based studies lack the amalgamation of the influence of geologic factors, specifically tectonics, for identifying groundwater fluoride distribution. This drawback encourages the present study to investigate the association of the tectonic framework with fluoride in a multi-model approach. We have applied three machine learning models (random forest, boosted regression tree, and logistic regression) to predict elevated groundwater fluoride based on fluoride measurements across India. The random forest model outperformed other models with an accuracy of 93%. Tectonics was found to be one of the most important predictors alongside "depth to water table." Two major areas of high risk identified were the northwest parts and the south-southeast cratonic peninsular region. The random forest model also performed significantly well over the validation dataset. We estimate that nearly 257 million people are exposed to elevated fluoride risk in India. We endeavor that the findings of our study would be an effective tool for identifying the areas at risk of elevated fluoride and also assist in undertaking effective groundwater management strategies.

Vulnerability of groundwater from elevated nitrate pollution across India: Insights from spatio-temporal patterns using large-scale monitoring data.

Agriculture-sourced, non-point groundwater contamination (e.g., nitrate) is a serious concern from the drinking water crisis aspect across the agrarian world. India is one of the largest consumers of nitrogen fertilizers in South-Asia as well as in the world but groundwater nitrate lacks critical attention as a wide-scale drinking water pollutant in the country. Our study provides the first documentation of the distribution of groundwater nitrate and the extent of elevated nitrate contamination across India, along with the delineation of the temporal trends and the natural and anthropogenic factors that influence such occurrence of groundwater nitrate. High resolution, annual-scale spatio-temporal variability of groundwater nitrate concentration and consequent contamination was delineated using groundwater nitrate measurements from ~3 million drinking water wells spread across 7038 administrative blocks between 2010 and 2017 in India. An average 8% of the studied blocks were found affected by elevated groundwater nitrate (> 45 mg/L). Depth-dependent trend demonstrated that nitrate concentrations were about 14% higher in shallow water wells (≤ 35 m) than deep wells (>35 m). The overall temporal trend of groundwater nitrate concentration was decreasing slightly nationwide in the study period. The correlation tests and causality test results indicated that the spatial distribution of groundwater nitrate was significantly associated with agricultural N-fertilizer usage, whereas the decreasing temporal trend corresponded with the overall reduced N-fertilizer usage during the study period. Spatial autocorrelation analysis identified the clustering of high nitrate areas in central, north, and southern India, specifically in areas with higher fertilizer usage. We estimate about 71 million Indians possibly exposed to elevated groundwater nitrate concentrations and the majority of them reside in rural areas. Thus, this study provides the previously unrecognized, wide-scale, anthropogenic, diffused groundwater nitrate contamination across India.

Occurrence, predictors and hazards of elevated groundwater arsenic across India through field observations and regional-scale AI-based modeling.

Existence of wide spread elevated concentrations of groundwater arsenic (As) across South Asia, including India, has endangered a huge groundwater-based drinking water dependent population. Here, using high-spatial resolution As field-observations (~3 million groundwater sources) across India, we have delineated the regional-scale occurrence of elevated groundwater As (≥10 µg/L), along with the possible geologic-geomorphologic-hydrologic and human-sourced predictors that influence the spatial distribution of the contaminant. Using statistical and machine learning method, we also modeled the groundwater As concentrations probability at 1 Km resolution, along with probabilistic delineation of high As-hazard zones across India. The observed occurrence of groundwater As was found to be most strongly influenced by geology-tectonics, groundwater-fed irrigated area (%) and elevation. Pervasive As contamination is observed in major parts of the Himalayan mega-river Indus-Ganges-Brahmaputra basins, however it also occurs in several more-localized pockets, mostly related to ancient tectonic zones, igneous provinces, aquifers in modern delta and chalcophile mineralized regions. The model results suggest As-hazard potential in yet-undetected areas. Our model performed well in predicting groundwater arsenic, with accuracy: 82% and 84%; area under the curve (AUC): 0.89 and 0.88 for test data and validation datasets. An estimated ~90 million people across India are found to be exposed to high groundwater As from field-observed data, with the five states with highest hazard are West Bengal (28 million), Bihar (21 million), Uttar Pradesh (15 million), Assam (8.6 million) and Punjab (6 million). However it can be much more if the modeled hazard is considered (>250 million). Thus, our study provides a detailed, quantitative assessment of high groundwater As across India, with delineation of possible intrinsic influences and exogenous forcings. The predictive model is helpful in predicting As-hazard zones in the areas with limited measurements.

Nanoparticle-complexed antimiRs for inhibiting tumor growth and metastasis in prostate carcinoma and melanoma.

BACKGROUND: MiRNAs act as negative regulators of gene expression through target mRNA degradation or inhibition of its translation. In cancer, several miRNAs are upregulated and play crucial roles in tumorigenesis, making the inhibition of these oncomiRs an interesting therapeutic approach. This can be achieved by directly complementary single-stranded anti-miRNA oligonucleotides (antimiRs). A major bottleneck in antimiR therapy, however, is their efficient delivery. The nanoparticle formation with polyethylenimine (PEI) may be particularly promising, based on the PEI's ability to electrostatically interact with oligonucleotides. This leads to their protection and supports delivery. In the present study, we explore for the first time PEI for antimiR formulation and delivery. We use the branched low molecular weight PEI F25-LMW for the complexation of different antimiRs, and analyse tumor- and metastasis-inhibitory effects of PEI/antimiR complexes in different tumor models. RESULTS: In prostate carcinoma, transfection of antimiRs against miR-375 and miR-141 leads to tumor cell inhibition in 2D- and 3D-models. More importantly, an in vivo tumor therapy study in prostate carcinoma xenografts reveals anti-tumor effects of the PEI/antimiR complexes. In advanced melanoma and metastasis, we identify by a microRNA screen miR-150 as a particularly relevant oncomiR candidate, and validate this result in vitro and in vivo. Again, the systemic application of PEI/antimiR complexes inhibiting this miRNA, or the previously described antimiR-638, leads to profound tumor growth inhibition. These effects are associated with the upregulation of direct miRNA target genes. In a melanoma metastasis mouse model, anti-metastatic effects of PEI/antimiR treatment are observed as well. CONCLUSIONS: We thus describe PEI-based complexes as efficient platform for antimiR therapy, as determined in two different tumor entities using in vivo models of tumor growth or metastasis. Our study also highlights the therapeutic relevance of miR-375, miR-141, miR-150 and miR-638 as target miRNAs for antimiR-mediated inhibition.

Modeling regional-scale groundwater arsenic hazard in the transboundary Ganges River Delta, India and Bangladesh: Infusing physically-based model with machine learning.

For the last few decades, toxic levels of arsenic (As) in groundwater from the aquifers of the Ganges River delta, India and Bangladesh, have been known to cause serious public health concerns. Innumerable studies have advocated the control of geomorphologic, geologic, hydrogeologic, biogeochemical, and anthropogenic factors on arsenic mobilization, flow, and distribution patterns within the Ganges River delta. We have developed transboundary regional-scale models for computing the probability of groundwater As concentrations to exceed the WHO permissible thresholds for drinking water of 10 µg/L within the Ganges River delta as a function of the various geomorphologic-(hydro)geologic-hydrostratigraphic-anthropogenic controlling factors, using statistical methods and artificial intelligence (AI) [i.e., machine learning] techniques namely, Random Forest (RF), Boosted Regression Trees (BRT) and Logistic Regression (LR) algorithms, followed by probabilistic delineation the high As-hazard zones within the delta. A "hybrid multi-modeling approach" was adapted for this study, which involved the introduction of hydrostratigraphic parameters (aquifer connectivity and surficial aquitard thickness) derived from a high-resolution transboundary hydrostratigraphic model developed for the Ganges River delta aquifer system, as predictors for modeling groundwater As probabilities within the delta. The RF model outperforms the BRT and LR model in terms of model performance. Model outputs suggest the dominant influence of surficial aquitard thickness and groundwater-fed irrigated area (%) on groundwater As. While, the north-central and southern regions of the Ganges River delta show low As-hazard (<10 µg/L), the western and north-eastern regions demonstrate elevated hazard level (>10 µg/L). An estimated 30.3 million people are found to be exposed to elevated groundwater As within the study area. Thus, our study demonstrates that such hybrid, predictive models are not only helpful in delineating the regional-scale distribution of groundwater As-hazard zones in the areas with limited As data but is also useful in identifying the possible exogenous forcing that may have led to the worst, natural pollution in human history.

H3K9me3-mediated epigenetic regulation of senescence in mice predicts outcome of lymphoma patients.

Lesion-based targeting strategies underlie cancer precision medicine. However, biological principles - such as cellular senescence - remain difficult to implement in molecularly informed treatment decisions. Functional analyses in syngeneic mouse models and cross-species validation in patient datasets might uncover clinically relevant genetics of biological response programs. Here, we show that chemotherapy-exposed primary Eµ-myc transgenic lymphomas - with and without defined genetic lesions - recapitulate molecular signatures of patients with diffuse large B-cell lymphoma (DLBCL). Importantly, we interrogate the murine lymphoma capacity to senesce and its epigenetic control via the histone H3 lysine 9 (H3K9)-methyltransferase Suv(ar)39h1 and H3K9me3-active demethylases by loss- and gain-of-function genetics, and an unbiased clinical trial-like approach. A mouse-derived senescence-indicating gene signature, termed "SUVARness", as well as high-level H3K9me3 lymphoma expression, predict favorable DLBCL patient outcome. Our data support the use of functional genetics in transgenic mouse models to incorporate basic biology knowledge into cancer precision medicine in the clinic.

Wide exposure of persistent organic pollutants (PoPs) in natural waters and sediments of the densely populated Western Bengal basin, India.

Drinking water stress in South Asia is now widely known as a global paradigm. Extensive geogenic groundwater pollution is known in this area for a long time, specifically in the densely populated (~40 million) Western Bengal basin (WBB) of the state of West Bengal, India. Though anthropogenic-sourced groundwater pollution has been long suspected, it has been only sporadically reported thus far. The present study provides one of the first documentation of widespread existence and distribution of persistent organic pollutants [PoPs, e.g. pesticide (2014-2016) and polycyclic aromatic hydrocarbons (PAHs) (2015)] in the Ganges river (32 locations) water and groundwater (235 locations) of the WBB. All locations were found to have at least one of the 40 detected pesticides [predominated by Atrazine (0.95-3.93 µg/L) and Malathion (150-9330 µg/L)], their derivatives [e.g. Malaoxon (410-1420 µg/L)] and/or 16 PAHs [e.g. Naphthalene (4.9-10.6 µg/L), Phenanthrene (3.32-6.61 µg/L)]. Atrazine and Malathion were found to have concentrations up to 46 times higher than the permissible limits. Similar to pesticides in water, most of the sediment samples investigated obtain Malathion (56-200 µg/kg), malaoxon (>900 µg/kg). Sediment samples collected from 10-20 cm to 20-30 cm depth showed total PAHs concentration of 2.02 and 1.95 µg/kg respectively. While herbicides were found to be more common in agricultural areas, insecticides and PAHs dominate in urban areas, suggesting land-use to be an important controlling factor. An estimated 53% of urban and 44% of rural residents (~20 million total residents, including those in cosmopolitan areas of Kolkata) are potentially exposed to PoPs pollution in drinking water, in addition to much ill-famed geogenic, groundwater arsenic pollution exposure known from this area.

Role of aquifer media in determining the fate of polycyclic aromatic hydrocarbons in the natural water and sediments along the lower Ganges river basin.

Groundwater-sourced drinking water quality in South Asia, specifically India, is extremely stressed, mostly from the presence of many pervasive and geogenic pollutants. The presence and behavior of anthropogenic pollutants like polycyclic aromatic hydrocarbons (PAHs) are poorly investigated on a regional or basin-wide scale. The present study provides one of the first documentation of the presence and behavior of PAH in the aquifer sediments in the Ganges river basin. Lower and medium molecular weight PAHs, e.g., naphthalene, phenanthrene, and fluoranthene were detected in 79, 36, and 13% of samples (n = 25). The PAH level in groundwater was approximately five times lower than river water. The sorption behavior of PAHs were studied in experiments in presence/absence of organic carbon and by simulating advective transport of low to medium molecular weight PAHs, e.g., naphthalene, phenanthrene, and fluoranthene in aquifer sediments collected from agricultural, peri-urban, and urban areas. Naphthalene and phenanthrene adsorbed on quartz and kaolinite, but not on clay minerals like kaolinite. Fluoranthene adsorbed more favorably on kaolinite. Numerical modeling of the advective transport of PAHs in aquifers suggest up to 25 times faster movement of pollutants from irrigation-induced pumping, indicating the strong control of hydraulics on the spatial distribution of PAHs in subsurface.

Author Correction: Impact of sanitation and socio-economy on groundwater fecal pollution and human health towards achieving sustainable development goals across India from ground-observations and satellite-derived nightlight.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Impact of sanitation and socio-economy on groundwater fecal pollution and human health towards achieving sustainable development goals across India from ground-observations and satellite-derived nightlight.

Globally, ~1 billion people, mostly residing in Africa and South Asia (e.g. India), still lack access to clean drinking water and sanitation. Resulting, unsafe disposal of fecal waste from open-defecation to nearby drinking water sources severely endanger public health. Until recently, India had a huge open-defecating population, leading declining public health from water-borne diseases like diarrhoea by ingesting polluted water, mostly sourced to groundwater. However, in recent past, sanitation development to achieve Sustainable Development Goals (SDGs) has been encouraged throughout India, but their effect to groundwater quality and human health conditions are yet-unquantified. Here, for the first time, using long term, high-spatial resolution measurements (>1.7 million) across India and analyses, we quantified that over the years, groundwater fecal coliform concentration (2002-2017, -2.56 ± 0.06%/year) and acute diarrheal cases (1990-2016, -3.05 ± 0.01%/year) have significantly reduced, potentially influenced by sanitation development (1990-2017, 2.63 ± 0.01%/year). Enhanced alleviation of groundwater quality and human health have been observed since 2014, with initiation of acceletated constructions of sanitation infrastructures through Clean India (Swachh Bharat) Mission. However, the goal of completely faecal-pollution free, clean drinking water is yet to be achieved. We also evaluated the suitability of using satellite-derived night-time light (NLan, 1992-2013, 4.26 ± 0.05%/year) as potential predictor for such economic development. We observed that in more than 80% of the study region, night-time light demonstrated to be a strong predictor for observed changes in groundwater quality, sanitation development and water-borne disease cases. While sanitation and economic development can improve public health, poor education level and improper human practices can strongly influence on water-borne diseases loads and thus health in parts of India.

Nontargeted Identification of Tracer Incorporation in High-Resolution Mass Spectrometry.

"Fluxomics" refers to the systematic analysis of metabolic fluxes in a biological system and may uncover novel dynamic properties of metabolism that remain undetected in conventional metabolomic approaches. In labeling experiments, tracer molecules are used to track changes in the isotopologue distribution of metabolites, which allows one to estimate fluxes in the metabolic network. Because unidentified compounds cannot be mapped on pathways, they are often neglected in labeling experiments. However, using recent developments in de novo annotation may allow to harvest the information present in these compounds if they can be identified. Here, we present a novel tool (HiResTEC) to detect tracer incorporation in high-resolution mass spectrometry data sets. The software automatically extracts a comprehensive, nonredundant list of all compounds showing more than 1% tracer incorporation in a nontargeted fashion. We explain and show in an example data set how mass precision and other filter heuristics, calculated on the raw data, can efficiently be used to reduce redundancy and noninformative signals by 95%. Ultimately, this allows to quickly investigate any labeling experiment for a complete set of labeled compounds (here 149) with acceptable false positive rates. We further re-evaluate a published data set from liquid chromatography-electrospray ionization (LC-ESI) to demonstrate broad applicability of our tool and emphasize importance of quality control (QC) tests. HiResTEC is provided as a package in the open source software framework R and is freely available on CRAN.

Water, sanitation and hygiene: The unfinished agenda in the World Health Organization South-East Asia Region.

Access to adequate water, sanitation and hygiene (WASH) is essential for the health, well-being and dignity of all people. The World Health Organization South-East Asia Region has made considerable progress in WASH provision during the past two decades. However, compared with increases in coverage of improved drinking water, in some parts of the region, access to adequate sanitation remains low, with continued prevalence of open defecation. The Sustainable Development Goals (SDGs) have set ambitious targets for WASH services to be achieved by 2030. Examples of major health outcomes that would benefit from meeting these targets are diarrhoea and nutrition status. Although the total number of deaths attributable to diarrhoea declined substantially between 1990 and 2012, inadequate WASH still accounts for more than 1000 child deaths each day worldwide. And, despite the reductions in mortality, diarrhoea morbidity attributable to diarrhoea remains unchanged at around 1.7 billion cases per year. It has been known for decades that repeated episodes of diarrhoea increase a child's risk of long-term undernutrition, reduced growth and impaired cognitive development. Nutritional effects of inadequate WASH also include environmental enteropathy, leading to chronic intestinal inflammation, malnutrition and developmental deficits in young children. Inadequate WASH also contributes to iron deficiency anaemia resulting from infestation with soil-transmitted helminths. The cross-sectoral emphasis of the SDGs should act as a stimulus for intersectoral collaboration on research and interventions to reduce all inequities that result from inadequate WASH.

The Effectiveness of Sanitary Inspections as a Risk Assessment Tool for Thermotolerant Coliform Bacteria Contamination of Rural Drinking Water: A Review of Data from West Bengal, India.

AbstractThe use of sanitary inspections combined with periodic water quality testing has been recommended in some cases as screening tools for fecal contamination. We conducted sanitary inspections and tested for thermotolerant coliforms (TTCs), a fecal indicator bacteria, among 7,317 unique water sources in West Bengal, India. Our results indicate that the sanitary inspection score has poor ability to identify TTC-contaminated sources. Among deep and shallow hand pumps, the area under curve (AUC) for prediction of TTC > 0 was 0.58 (95% confidence interval [CI] = 0.53-0.61) and 0.58 (95% CI = 0.54-0.62), respectively, indicating that the sanitary inspection score was only marginally better than chance in discriminating between contaminated and uncontaminated sources of this type. A slightly higher AUC value of 0.64 (95% CI=0.57-0.71) was observed when the sanitary inspection score was used for prediction of TTC > 0 among the gravity-fed piped sources. Among unprotected springs (AUC = 0.48, 95% CI = 0.38-0.55) and unprotected dug wells (AUC = 0.41, 95% CI = 0.20-0.66), the sanitary inspection score performed more poorly than chance in discriminating between sites with TTC < 1 and TTC > 0. Aggregating over all source types, the sensitivity (true positive rate) of a high/very high sanitary inspection score for TTC contamination (TTC > 1 CFU/100 mL) was 29.4% and the specificity (true negative rate) was 77.9%, resulting in substantial misclassification of the sites when using the established risk categories. These findings suggest that sanitary surveys are inappropriate screening tools for identifying TTC contamination at water points.

A mutation in the NADH-dehydrogenase subunit 2 suppresses fibroblast aging.

Mutations of mitochondrial (mt)DNA cause a variety of human diseases and are implicated in premature aging syndromes. Here we investigated a single nucleotide exchange (leucine to methionine) at position nt4738 in the mitochondrial NADH dehydrogenase subunit 2 (Nd2) gene of the respiratory chain. Primary fibroblasts derived from the conplastic mouse strain C57BL/6J-mtALR/LTJ with mutant enzyme, possessed high enzyme activity and ATP production and low ROS production. Furthermore, Nd2-mutant fibroblasts expressed lower senescence markers. Transcriptome analysis revealed that the members of the p38MAPK pathway were significantly downregulated in Nd2-mutant mice. In agreement, inhibition of p38MAPK with SB203580 enhanced proliferation and reduced cytokine secretion in fibroblasts. In Nd2-mutant mouse skin, the amount of Ki67-positive cells was significantly higher than in control skin. The higher amount of Ki67-positive cells and the thicker epidermis in Nd2-mutant mice strongly supported the in vitro data. In conclusion, Nd2 is a mitochondrial gene, involved in age-related signaling pathways.

miR-638 promotes melanoma metastasis and protects melanoma cells from apoptosis and autophagy.

The present study identified miR-638 as one of the most significantly overexpressed miRNAs in metastatic lesions of melanomas compared with primary melanomas. miR-638 enhanced the tumorigenic properties of melanoma cells in vitro and lung colonization in vivo. mRNA expression profiling identified new candidate genes including TP53INP2 as miR-638 targets, the majority of which are involved in p53 signalling. Overexpression of TP53INP2 severely attenuated proliferative and invasive capacity of melanoma cells which was reversed by miR-638. Depletion of miR-638 stimulated expression of p53 and p53 downstream target genes and induced apoptosis and autophagy. miR-638 promoter analysis identified the miR-638 target transcription factor associated protein 2α (TFAP2A/AP-2α) as a direct negative regulator of miR-638, suggestive for a double-negative regulatory feedback loop. Taken together, miR-638 supports melanoma progression and suppresses p53-mediated apoptosis pathways, autophagy and expression of the transcriptional repressor TFAP2A/AP-2α.

Chk1 and Wee1 control genotoxic-stress induced G2-M arrest in melanoma cells.

In the present report, the role of ATR-Chk1-Wee1 and ATM-Chk2-p53-p21 pathways in stress-induced cell cycle control is analysed in melanoma cells. Treatment of p53 wild-type melanoma cells with the genotoxic agent doxorubicin induces G2-M arrest, inhibitory phosphorylation of cell cycle kinase Cdc2 (CDK1) and enhanced expression of p53/p21. Wee1 inhibition under doxorubicin pulse-treatment reduces G2-M arrest and induces apoptosis. Inhibition of upstream kinase Chk1 under doxorubicin treatment almost completely abolishes stress-induced G2-M arrest and induces enhanced apoptosis. Interestingly, Chk1 inhibition alone even further increases apoptosis. While Chk1 inhibition alone almost completely abolishes G0-G1 arrest, combined treatment with doxorubicin re-establishes G0-G1 arrest. Moreover, Chk1 inhibition alone induces only a slight p53/p21 induction, while a strong induction of both proteins is observed by the combination with doxorubicin. These findings are suggestive for a particular role of p53/p21 in G0-G1, and Chk1 in G0-G1 and G2-M arrest. In line with this, the p53-mutant SK-Mel-28 melanoma cells do not mount a significant G0-G1 arrest under combined doxorubicin and Chk1 inhibitor treatment but rather show extensive apoptosis. Moreover, knockdown of p21 dramatically reduces stress-induced G0-G1 arrest under doxorubicin and Chk1 inhibitor treatment accompanied by massive DNA damage and apoptosis induction. Treatment of melanoma cells with an inhibitor of Chk2 upstream kinase ATM and doxorubicin almost completely abolishes G0-G1 arrest. Taken together, both Chk1 and Wee1 are mediators of G2-M arrest, while p53, p21 and Chk1 are mediators of G0-G1 arrest in melanoma cells. Combined treatment with chemotherapeutic agents such as doxorubicin and Chk1 inhibitors may help to overcome apoptosis resistance of p53-proficient melanoma cells. But treatment with Chk1 inhibitor alone may even be more efficient.

Genomewide RNAi screen identifies protein kinase Cb and new members of mitogen-activated protein kinase pathway as regulators of melanoma cell growth and metastasis.

A large-scale RNAi screen was performed for eight different melanoma cell lines using a pooled whole-genome lentiviral shRNA library. shRNAs affecting proliferation of transduced melanoma cells were negatively selected during 10 days of culture. Overall, 617 shRNAs were identified by microarray hybridization. Pathway analyses identified mitogen-activated protein kinase (MAPK) pathway members such as ERK1/2, JNK1/2 and MAP3K7 and protein kinase C ß (PKCß) as candidate genes. Knockdown of PKCß most consistently reduced cellular proliferation, colony formation and migratory capacity of melanoma cells and was selected for further validation. PKCß showed enhanced expression in human primary melanomas and distant metastases as compared with benign melanocytic nevi. Moreover, treatment of melanoma cells with PKCß-specific inhibitor enzastaurin reduced melanoma cell growth but had only small effects on benign fibroblasts. Finally, PKCß-shRNA significantly reduced lung colonization capacity of stably transduced melanoma cells in mice. Taken together, this study identified new candidate genes for melanoma cell growth and proliferation. PKCß seems to play an important role in these processes and might serve as a new target for the treatment of metastatic melanoma.

A systems' biology approach to study microRNA-mediated gene regulatory networks.

MicroRNAs (miRNAs) are potent effectors in gene regulatory networks where aberrant miRNA expression can contribute to human diseases such as cancer. For a better understanding of the regulatory role of miRNAs in coordinating gene expression, we here present a systems biology approach combining data-driven modeling and model-driven experiments. Such an approach is characterized by an iterative process, including biological data acquisition and integration, network construction, mathematical modeling and experimental validation. To demonstrate the application of this approach, we adopt it to investigate mechanisms of collective repression on p21 by multiple miRNAs. We first construct a p21 regulatory network based on data from the literature and further expand it using algorithms that predict molecular interactions. Based on the network structure, a detailed mechanistic model is established and its parameter values are determined using data. Finally, the calibrated model is used to study the effect of different miRNA expression profiles and cooperative target regulation on p21 expression levels in different biological contexts.