Prioritization of cancer therapeutic targets using CRISPR-Cas9 screens.

Functional genomics approaches can overcome limitations-such as the lack of identification of robust targets and poor clinical efficacy-that hamper cancer drug development. Here we performed genome-scale CRISPR-Cas9 screens in 324 human cancer cell lines from 30 cancer types and developed a data-driven framework to prioritize candidates for cancer therapeutics. We integrated cell fitness effects with genomic biomarkers and target tractability for drug development to systematically prioritize new targets in defined tissues and genotypes. We verified one of our most promising dependencies, the Werner syndrome ATP-dependent helicase, as a synthetic lethal target in tumours from multiple cancer types with microsatellite instability. Our analysis provides a resource of cancer dependencies, generates a framework to prioritize cancer drug targets and suggests specific new targets. The principles described in this study can inform the initial stages of drug development by contributing to a new, diverse and more effective portfolio of cancer drug targets.

Identifying Informal Settlements Using Contourlet Assisted Deep Learning.

As the global urban population grows due to the influx of migrants from rural areas, many cities in developing countries face the emergence and proliferation of unplanned and informal settlements. However, even though the rise of unplanned development influences planning and management of residential land-use, reliable and detailed information about these areas is often scarce. While formal settlements in urban areas are easily mapped due to their distinct features, this does not hold true for informal settlements because of their microstructure, instability, and variability of shape and texture. Therefore, detecting and mapping these areas remains a challenging task. This research will contribute to the development of tools to identify such informal built-up areas by using an integrated approach of multiscale deep learning. The authors propose a composite architecture for semantic segmentation using the U-net architecture aided by information obtained from a multiscale contourlet transform. This work also analyzes the effects of wavelet and contourlet decompositions in the U-net architecture. The performance was evaluated in terms of precision, recall, F-score, mean intersection over union, and overall accuracy. It was found that the proposed method has better class-discriminating power as compared to existing methods and has an overall classification accuracy of 94.9-95.7%.

Unsupervised correction of gene-independent cell responses to CRISPR-Cas9 targeting.

BACKGROUND: Genome editing by CRISPR-Cas9 technology allows large-scale screening of gene essentiality in cancer. A confounding factor when interpreting CRISPR-Cas9 screens is the high false-positive rate in detecting essential genes within copy number amplified regions of the genome. We have developed the computational tool CRISPRcleanR which is capable of identifying and correcting gene-independent responses to CRISPR-Cas9 targeting. CRISPRcleanR uses an unsupervised approach based on the segmentation of single-guide RNA fold change values across the genome, without making any assumption about the copy number status of the targeted genes. RESULTS: Applying our method to existing and newly generated genome-wide essentiality profiles from 15 cancer cell lines, we demonstrate that CRISPRcleanR reduces false positives when calling essential genes, correcting biases within and outside of amplified regions, while maintaining true positive rates. Established cancer dependencies and essentiality signals of amplified cancer driver genes are detectable post-correction. CRISPRcleanR reports sgRNA fold changes and normalised read counts, is therefore compatible with downstream analysis tools, and works with multiple sgRNA libraries. CONCLUSIONS: CRISPRcleanR is a versatile open-source tool for the analysis of CRISPR-Cas9 knockout screens to identify essential genes.

Unravelling cell type-specific responses to Parkinson's Disease at single cell resolution.

Parkinson's Disease (PD) is the second most common neurodegenerative disorder. The pathological hallmark of PD is loss of dopaminergic neurons and the presence of aggregated α-synuclein, primarily in the substantia nigra pars compacta (SNpc) of the midbrain. However, the molecular mechanisms that underlie the pathology in different cell types is not currently understood. Here, we present a single nucleus transcriptome analysis of human post-mortem SNpc obtained from 15 sporadic Parkinson's Disease (PD) cases and 14 Controls. Our dataset comprises ∼84K nuclei, representing all major cell types of the brain, allowing us to obtain a transcriptome-level characterization of these cell types. Importantly, we identify multiple subpopulations for each cell type and describe specific gene sets that provide insights into the differing roles of these subpopulations. Our findings reveal a significant decrease in neuronal cells in PD samples, accompanied by an increase in glial cells and T cells. Subpopulation analyses demonstrate a significant depletion of tyrosine hydroxylase (TH) enriched astrocyte, microglia and oligodendrocyte populations in PD samples, as well as TH enriched neurons, which are also depleted. Moreover, marker gene analysis of the depleted subpopulations identified 28 overlapping genes, including those associated with dopamine metabolism (e.g., ALDH1A1, SLC6A3 & SLC18A2). Overall, our study provides a valuable resource for understanding the molecular mechanisms involved in dopaminergic neuron degeneration and glial responses in PD, highlighting the existence of novel subpopulations and cell type-specific gene sets.

Single-cell transcriptomics identifies perturbed molecular pathways in midbrain organoids using α-synuclein triplication Parkinson's disease patient-derived iPSCs.

Three-dimensional (3D) brain organoids provide a platform to study brain development, cellular coordination, and disease using human tissue. Here, we generate midbrain dopaminergic (mDA) organoids from induced pluripotent stem cells (iPSC) from healthy and Parkinson's Disease (PD) donors and assess them as a human PD model using single-cell RNAseq. We characterize cell types in our organoid cultures and analyze our model's Dopamine (DA) neurons using cytotoxic and genetic stressors. Our study provides the first in-depth, single-cell analysis of SNCA triplication and shows evidence for molecular dysfunction in oxidative phosphorylation, translation, and ER protein-folding in DA neurons. We perform an in-silico identification of rotenone-sensitive DA neurons and characterization of corresponding transcriptomic profiles associated with synaptic signalling and cholesterol biosynthesis. Finally, we show a novel chimera organoid model from healthy and PD iPSCs allowing the study of DA neurons from different individuals within the same tissue.

CRISPR-Cas9 genetic screen leads to the discovery of L-Moses, a KAT2B inhibitor that attenuates Tunicamycin-mediated neuronal cell death.

Accumulation of aggregated and misfolded proteins, leading to endoplasmic reticulum stress and activation of the unfolded protein response, is a hallmark of several neurodegenerative disorders, including Alzheimer's and Parkinson's disease. Genetic screens are powerful tools that are proving invaluable in identifying novel modulators of disease associated processes. Here, we performed a loss-of-function genetic screen using a human druggable genome library, followed by an arrayed-screen validation, in human iPSC-derived cortical neurons. We identified and genetically validated 13 genes, whose knockout was neuroprotective against Tunicamycin, a glycoprotein synthesis inhibitor widely used to induce endoplasmic reticulum stress. We also demonstrated that pharmacological inhibition of KAT2B, a lysine acetyltransferase identified by our genetic screens, by L-Moses, attenuates Tunicamycin-mediated neuronal cell death and activation of CHOP, a key pro-apoptotic member of the unfolded protein response in both cortical and dopaminergic neurons. Follow-up transcriptional analysis suggested that L-Moses provided neuroprotection by partly reversing the transcriptional changes caused by Tunicamycin. Finally, L-Moses treatment attenuated total protein levels affected by Tunicamycin, without affecting their acetylation profile. In summary, using an unbiased approach, we identified KAT2B and its inhibitor, L-Moses, as potential therapeutic targets for neurodegenerative diseases.

A suspension technique for efficient large-scale cancer organoid culturing and perturbation screens.

Organoid cell culture methodologies are enabling the generation of cell models from healthy and diseased tissue. Patient-derived cancer organoids that recapitulate the genetic and histopathological diversity of patient tumours are being systematically generated, providing an opportunity to investigate new cancer biology and therapeutic approaches. The use of organoid cultures for many applications, including genetic and chemical perturbation screens, is limited due to the technical demands and cost associated with their handling and propagation. Here we report and benchmark a suspension culture technique for cancer organoids which allows for the expansion of models to tens of millions of cells with increased efficiency in comparison to standard organoid culturing protocols. Using whole-genome DNA and RNA sequencing analyses, as well as medium-throughput drug sensitivity testing and genome-wide CRISPR-Cas9 screening, we demonstrate that cancer organoids grown as a suspension culture are genetically and phenotypically similar to their counterparts grown in standard conditions. This culture technique simplifies organoid cell culture and extends the range of organoid applications, including for routine use in large-scale perturbation screens.

Nordihydroguaiaretic Acid from Creosote Bush (Larrea tridentata) Mitigates 12-O-Tetradecanoylphorbol-13-Acetate-Induced Inflammatory and Oxidative Stress Responses of Tumor Promotion Cascade in Mouse Skin.

Nordihydroguaiaretic acid (NDGA) is a phenolic antioxidant found in the leaves and twigs of the evergreen desert shrub, Larrea tridentata (Sesse and Moc. ex DC) Coville (creosote bush). It has a long history of traditional medicinal use by the Native Americans and Mexicans. The modulatory effects of topically applied NDGA was studied on acute inflammatory and oxidative stress responses in mouse skin induced by stage I tumor promoting agent, 12-O-tetradecanoylphorbol-13-acetate (TPA). Double TPA treatment adversely altered many of the marker responses of stage I skin tumor promotion cascade. Pretreatment of NDGA in TPA-treated mice mitigated cutaneous lipid peroxidation and inhibited production of hydrogen peroxide. NDGA treatment also restored reduced glutathione level and activities of antioxidant enzymes. Elevated activities of myeloperoxidase, xanthine oxidase and skin edema formation in TPA-treated mice were also lowered by NDGA indicating a restrained inflammatory response. Furthermore, results of histological study demonstrated inhibitory effect of NDGA on cellular inflammatory responses. This study provides a direct evidence of antioxidative and anti-inflammatory properties of NDGA against TPA-induced cutaneous inflammation and oxidative stress corroborating its chemopreventive potential against skin cancer.

In vivo cytogenetic and oxidative stress-inducing effects of cypermethrin in freshwater fish, Channa punctata Bloch.

Synthetic pyrethroids are considered to be safe over other insecticides. Many of the newest pyrethroids lack ecotoxicity data. However, animal data indicate that their use may pose risk to environmental biota. The cytogenetic effects of cypermethrin, an α-class type II pyrethroid were evaluated using robust genotoxicity assay of chromosomal aberration (CA) and micronucleus (MN) tests in highly mitotic kidney cells and in erythrocytes of a freshwater fish, Channa punctata Bloch. In order to understand biochemical mechanism of genotoxic effects, oxidative stress parameters were also studied in fish erythrocytes. Fish exposed to cypermethrin (0.4, 0.8 and 1.2 µg/l for 48 and 72 h) showed increased frequencies of CA and MN in a concentration-dependent manner. Fish exposed to positive genotoxin, ethyl methane sulfonate (EMS) also showed significant increase in frequencies of CA and MN. The genotoxic effects were invariably accompanied and correlated with increased oxidative stress and disturbance of antioxidant enzymes.

Azotobacter: A potential bio-fertilizer for soil and plant health management.

Stressor (biotic as well as abiotic) generally hijack the plant growth and yield characters in hostile environment leading to poor germination of the plants and yield. Among the plant growth promoting rhizobacteria, Azotobacter spp. (Gram-negative prokaryote) are considered to improve the plant health. Various mechanisms are implicated behind improved plant health in Azotobacter spp. inoculated plants. For example, acceleration of phytohormone like Indole-3-Acetic Acid production, obviation of various stressors, nitrogen fixation, pesticides and oil globules degradation, heavy metals metabolization, etc. are the key characteristics of Azotobacter spp. action. In addition, application of this bacteria has also become helpful in the reclamation of soil suggesting to be a putative agent which can be used in the transformation of virgin land to fertile one. Application of pesticides of chemical origin are being put on suspension mode as the related awareness program is still on. As far as the limitations of this microbe is concerned, commercial level formulations availability is still a great menace. Present review has been aimed to appraise the researchers pertaining to utility of Azotobacter spp. in the amelioration of plant health in sustainable agroecosystem. The article has been written with the target to gather maximum information into single pot so that it could reach to the dedicated researchers.

Iron deficiency augments bisphenol A-induced oxidative stress in rats.

Bisphenol A (BPA), an estrogenic environmental contaminant is also known for oxidative stress-inducing effect. Malnutrition is recognized as a confounding factor in oxidative stress. However, little is known about effect of malnutrition on oxidative stress induced by BPA or other endocrine disrupting chemicals (EDCs). We studied effect of malnutrition (iron deficiency) in rats chronically exposed to low levels of BPA taking into consideration the oxidative stress and antioxidant status in liver, kidney and gonads. Iron deficiency significantly elevated level of lipid peroxidation in BPA-exposed rats. Similarly, decrease in reduced glutathione level was more significant in rats maintained on iron deficient diet compared to those maintained on normal diet. Iron deficiency also significantly modulated activities of vital antioxidant enzymes in all the tissues. Female rats showed more vulnerability than males to iron-deficiency modulated effects of BPA on the above parameters. This study demonstrated that malnutrition, especially iron deficiency, might act as a confounding factor in EDC-induced oxidative stress. However, more studies may be needed to confirm effect of nutritional factors on estrogenic activity of BPA or other EDCs.

Genotoxic and oxidative stress-inducing effects of deltamethrin in the erythrocytes of a freshwater biomarker fish species, Channa punctata Bloch.

Deltamethrin, an alpha-cyano class of pyrethroid insecticide is used in insect pest control and antimalaria programs in several countries including India. Although various toxic manifestations of deltamethrin are reported in mammals, its ecotoxicologic dimensions are not adequately researched in ecologically and commercially important fishes. In this study, we report genotoxic effect of deltamethrin in a biomarker fish Channa punctata (Bloch). Adult fish were exposed to three concentrations of technical grade deltamethrin (0.4, 0.8, and 1.2 microg/L) for 48 and 72 h. Ethyl methane sulfonate was used as a positive control. Fish were analyzed for induction of micronucleus (MN), nuclear abnormalities (NAs), and oxidative stress biomarkers in erythrocytes. Deltamethrin significantly induced MN and NAs accompanied by increased lipid peroxidation. Activity of antioxidant enzyme superoxide dismutase was significantly decreased but an increase was observed in reduced glutathione level after 72 h of exposure. The NAs in exposed fish included blebbed, lobed and notched nuclei, and binucleated erythrocytes. Our findings suggest that oxidative stress may, in part, be contributing to deltamethrin-induced genotoxic damage to erythrocytes. Although MN induction is a nonspecific biomarker, it may provide an indication of pollution load of deltamethrin in the affected fish population when used as part of suite of other biomarkers.

Incidence Of Paraphenylene-Diamine Poisoning In Three Districts Headquarter Hospitals Of Pakistan.

BACKGROUND: Paraphenylene-diamine (PPD) poisoning is an emerging problem of developing African and South Asian countries. This study was done with the objective to determine the clinical lab diagnostic accuracy of serum creatinine phosphokinase in cases initially reporting facial oedema followed by renal failure and rhabdomyolysis due to paraphenylene-diamine (PPD) poisoning. METHODS: It was a cross-sectional study in which data was retrospectively collected at District Head Quarter (DHQ) Hospitals. Data was collected over a period of one year from Jan- Dec 2018. Data was collected from the patient file records. Data was analysed on SPSS version 20. RESULTS: In the present study, 658 cases of Kala Pathar poisoning presented and treated. M: F ratio was 5:20. There were 518 (78.8%) females. Majority of the female patients were married 488 (94%). Most common clinical manifestations included marked facial oedema; dysphagia and stridor. Post complications include rhabdomyolysis and acute renal failure which developed after two to five days. Initial lab investigations within 6-8 hours after ingestion showed marked increase in TLC count, ALT and Na+ ions. There was marked elevation of serum CPK (1400±200 U/L) levels after 24 hours. CONCLUSIONS: PPD poisoning is more common in females of younger age group belonging to rural areas. Early diagnosis and prompt supportive treatment within 2-12 hours of ingestion can save many lives. There is no specific antidote available for this poison.

Effects of exposure to multiple trace metals on biochemical, histological and ultrastructural features of gills of a freshwater fish, Channa punctata Bloch.

The trace metals are frequently encountered as mixtures of essential and non-essential elements. Therefore, evaluation of their toxic effects individually does not offer a realistic estimate of their impact on biological processes. We studied effects of a mixture of four essential and toxic metals (Cu, Cd, Fe and Ni) on biochemical and morphological characteristics of the gills of a biomarker freshwater fish Channa punctata (Bloch) using environmentally relevant concentrations. Fish were exposed to metal mixture through tank water for 7, 15 and 30 days. Biochemical studies as well as light microscopy (LM) and scanning electron microscopy (SEM) revealed significant metal exposure-induced alterations in gills. Besides ultastructural changes, activities of antioxidant enzymes such catalase (CAT), glutathione S-transferase (GST) and superoxide dismutase (SOD) were significantly altered in the gills of exposed fish. The reduced glutathione (GSH) was significantly (p<0.001) decreased, while lipid peroxidation (LPO) was significantly (p<0.001) increased. The main alterations in general morphology of fish gills included spiking and fusion of secondary lamellae, formation of club-shaped filaments, and vacuolization and necrosis of filament epithelium in the interlamellar regions. SEM studies showed gradual increase of the density and apical surface area of the chloride cells and transformation of the surface structure of the pavement cells. The results of this study indicate adaptive as well a toxic responses in fish gills exposed to mixture of trace metals. Low concentrations of trace metal appear to compromise the antioxidant defense of gills. Lesions in the gill morphology caused by the effect of low concentrations of trace metals could lead to functional alterations and interference with fundamental processes such as maintenance of osmoregulation, gas exchange and xenobiotic metabolism in the exposed fish populations.

Channa punctata brain metallothionein is a potent scavenger of superoxide radicals and prevents hydroxyl radical-induced in vitro DNA damage.

Mammalian brain metallothioneins (MTs) have been shown to scavenge free radicals. However, a similar role for fish brain MT has not been established yet. Previously, we have reported that MT from the liver of a freshwater fish, Channa punctata Bloch, had free-radical-scavenging activity in vitro. In this study, we report on the induction of MT in brain and other tissues of C. punctata treated with a low concentration of zinc chloride. We partially purified MT (Zn-MT)-rich fraction from the brain and studied its free-radical-scavenging and DNA damage attenuating effects. Zinc exposure showed significant MT induction in brain, gill, kidney, and liver. C. punctata brain MT efficiently scavenged superoxide radicals and also attenuated hydroxyl radical-mediated DNA damage. These findings suggest that fish brain MT has a free-radical-scavenging activity, and its expression may be regulated in response to stress and chemical exposure. C. punctata has been identified as a potent biomarker fish species. It is suggested that this fish species may be a good model for the study of MTs with regard to their regulatory and biomarker functions.

Pro-apoptotic effect of fly ash leachates in hepatocytes of freshwater fish (Channa punctata Bloch).

The pro-apoptotic effect of fly ash leachates (FAL) was studied in the hepatocytes of an Indian freshwater fish, Channa punctata Bloch. Hepatocytes were exposed to different concentrations of '7-day' FAL for 24 and 48h and various parameters of apoptosis were studied using standardized procedures. FAL-induced apoptosis in hepatocytes was indicated by cytological examination, DNA fragmentation and DNA laddering. The induction in cytochrome-c release, caspases 3, 7, 10 and 9 activities and lactate dehydrogenase level provide mechanistic platform for FAL-induced apoptosis. Cytological examination showed an unambiguous apoptotic effect of ash leachates in fish hepatocytes. Exposed hepatocytes also showed increased production of H(2)O(2), superoxide ions and an increase in lipid peroxidation (LPO). The present study suggests a possible role of reactive oxygen species (ROS) in FAL-induced apoptosis in hepatocytes. Lactate dehydrogenase, LPO and apoptosis as biomarkers of cytotoxicity have recently been used for assessment of ecotoxicological impact of environmental chemicals. Our findings show that these biomarkers may also be used for evaluation of ecotoxicological impact of complex chemical mixture such as fly ash and its leachates.

Successful Generation of Human Induced Pluripotent Stem Cell Lines from Blood Samples Held at Room Temperature for up to 48 hr.

The collection sites of human primary tissue samples and the receiving laboratories, where the human induced pluripotent stem cells (hIPSCs) are derived, are often not on the same site. Thus, the stability of samples prior to derivation constrains the distance between the collection site and the receiving laboratory. To investigate sample stability, we collected blood and held it at room temperature for 5, 24, or 48 hr before isolating peripheral blood mononuclear cells (PBMCs) and reprogramming into IPSCs. Additionally, PBMC samples at 5- and 48-hr time points were frozen in liquid nitrogen for 4 months and reprogrammed into IPSCs. hIPSC lines derived from all time points were pluripotent, displayed no marked difference in chromosomal aberration rates, and differentiated into three germ layers. Reprogramming efficiency at 24- and 48-hr time points was 3- and 10-fold lower, respectively, than at 5 hr; the freeze-thaw process of PBMCs resulted in no obvious change in reprogramming efficiency.

Nordihydroguiaretic acid attenuates skin tumorigenesis in Swiss albino mice with the condition of topical co-administration of an immunosuppressant.

Drug and chemically-induced immunosuppression has been implicated as a confounding factor for cancer development. Management of cancer in such situation is often a challenging task. We tested the efficacy of nordihydroguiaretic acid (NDGA) against immunosuppressant tacrolimus-induced augmentation of mouse skin tumorigenesis. It was observed that topical administration of tacrolimus significantly accelerated the tumor promotion events in dimethylbenz(a)anthracene (DMBA)-initiated and 12-O-tetradecanoylphorbol-13-acetate (TPA) promoted two-stage mouse skin carcinogenesis, which were accompanied by reduced CD4(+)/CD8(+) ratio of lymph nodes and serum IL-2 level. NDGA pre-treatment before each TPA application reduced the tumor incidence, its multiplicity and volume together with improvement in histopathological alterations and decrease in proliferating cell nuclear antigen (PCNA) labeling index (LI). However, NDGA had no significant influence on the immunosuppressive effect of tacrolimus. The present study demonstrates chemopreventive effect of NDGA in normal as well as in the condition of immunosuppression. Thus, NDGA has the potential to inhibit or delay the onset of tumor development during immunosuppressive regimen.

The interactive effect of elevated temperature on deltamethrin-induced biochemical stress responses in Channa punctata Bloch.

There are reports showing interactive effect of environmental factors with the toxic outcome of chemicals. We studied the interactive effect of elevated temperature as an abiotic stressor on deltamethrin-induced biochemical stress responses in a freshwater fish, Channa punctata Bloch. Heat stress (∼12°C above ambient temperature for 3h) and pesticide exposure (deltamethrin 0.75ppb for 48h) showed significant induction of heat shock protein-70 (HSP70) in liver, kidney and gills of fishes. Elevated temperature when followed by deltamethrin exposure showed synergistic effect showing a high level of HSP70 in liver and gills whereas response in the kidney was opposite. On the contrary, when deltamethrin exposure followed the heat stress, no significant difference was observed. Protein carbonylation was found to be more pronounced in heat-stressed group compared with control fish group. A significant increase in lipid peroxidation (LPO) was observed in different tissues of fish exposed to either of the stressors. In the kidney of fish exposed to heat stress followed by deltamethrin, LPO was relatively lower as compared to other treatments. Thiols content such as reduced glutathione (GSH), total thiols (T-SH), non-protein thiols (NP-SH) and protein thiols (P-SH) showed no consistent pattern in different tissues. In deltamethrin-exposed group that was subsequently exposed to heat stress, the GSH content was higher in liver and lower in both kidney and gills when compared with other groups. Alteration in the activities of antioxidant enzymes such as catalase (CAT), glutathione S-transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GPx) was also observed when fish were exposed to heat stress and/or deltamethrin. Our study demonstrated that heat stress modulated biochemical stress responses in fish showing a tissue specific pattern. This implies that fish has the capacity to elicit differential response to exposure to abiotic stressors in order to reduce the systemic magnitude of stress which may otherwise lead to severe dysfunction of vital tissues.