Transcriptional effects of carbon and nitrogen starvation on Ganoderma boninense, an oil palm phytopathogen.

BACKGROUND: Ganoderma boninense is a phytopathogen of oil palm, causing basal and upper stem rot diseases. METHODS: The genome sequence was used as a reference to study gene expression during growth in a starved carbon (C) and nitrogen (N) environment with minimal sugar and sawdust as initial energy sources. This study was conducted to mimic possible limitations of the C-N nutrient sources during the growth of G. boninense in oil palm plantations. RESULTS: Genome sequencing of an isolate collected from a palm tree in West Malaysia generated an assembly of 67.12 Mb encoding 19,851 predicted genes. Transcriptomic analysis from a time course experiment during growth in this starvation media identified differentially expressed genes (DEGs) that were found to be associated with 29 metabolic pathways. During the active growth phase, 26 DEGs were related to four pathways, including secondary metabolite biosynthesis, carbohydrate metabolism, glycan metabolism and mycotoxin biosynthesis. G. boninense genes involved in the carbohydrate metabolism pathway that contribute to the degradation of plant cell walls were up-regulated. Interestingly, several genes associated with the mycotoxin biosynthesis pathway were identified as playing a possible role in pathogen-host interaction. In addition, metabolomics analysis revealed six metabolites, maltose, xylobiose, glucooligosaccharide, glycylproline, dimethylfumaric acid and arabitol that were up-regulated on Day2 of the time course experiment. CONCLUSIONS: This study provides information on genes expressed by G. boninense in metabolic pathways that may play a role in the initial infection of the host.

Mast-Cell Tryptase Release Contributes to Disease Progression in Lymphangioleiomyomatosis.

Rationale: Lymphangioleiomyomatosis (LAM) is a multisystem disease that causes lung cysts and respiratory failure. Loss of TSC (tuberous sclerosis complex) gene function results in a clone of "LAM cells" with dysregulated mTOR (mechanistic target of rapamycin) activity. LAM cells and fibroblasts form lung nodules that also contain mast cells, although their significance is unknown. Objectives: To understand the mechanism of mast-cell accumulation and the role of mast cells in the pathogenesis of LAM. Methods: Gene expression was examined using transcriptional profiling and qRT-PCR. Mast cell/LAM nodule interactions were examined in vitro using spheroid TSC2-null cell/fibroblast cocultures and in vivo using an immunocompetent Tsc2-null murine homograft model. Measurements and Main Results: LAM-derived cell/fibroblast cocultures induced multiple CXC chemokines in fibroblasts. LAM lungs had increased tryptase-positive mast cells expressing CXCRs (CXC chemokine receptors) (P < 0.05). Mast cells located around the periphery of LAM nodules were positively associated with the rate of lung function loss (P = 0.016). LAM spheroids attracted mast cells, and this process was inhibited by pharmacologic and CRISPR/cas9 inhibition of CXCR1 and CXCR2. LAM spheroids caused mast-cell tryptase release, which induced fibroblast proliferation and increased LAM-spheroid size (1.36 ± 0.24-fold; P = 0.0019). The tryptase inhibitor APC366 and sodium cromoglycate (SCG) inhibited mast cell-induced spheroid growth. In vivo, SCG reduced mast-cell activation and Tsc2-null lung tumor burden (vehicle: 32.5.3% ± 23.6%; SCG: 5.5% ± 4.3%; P = 0.0035). Conclusions: LAM-cell/fibroblast interactions attract mast cells where tryptase release contributes to disease progression. Repurposing SCG for use in LAM should be studied as an alternative or adjunct to mTOR inhibitor therapy.

Brassinosteroids participate in the control of basal and acquired freezing tolerance of plants.

Brassinosteroids (BRs) are growth-promoting plant hormones that play a role in abiotic stress responses, but molecular modes that enable this activity remain largely unknown. Here we show that BRs participate in the regulation of freezing tolerance. BR signaling-defective mutants of Arabidopsis thaliana were hypersensitive to freezing before and after cold acclimation. The constitutive activation of BR signaling, in contrast, enhanced freezing resistance. Evidence is provided that the BR-controlled basic helix-loop-helix transcription factor CESTA (CES) can contribute to the constitutive expression of the C-REPEAT/DEHYDRATION-RESPONSIVE ELEMENT BINDING FACTOR (CBF) transcriptional regulators that control cold responsive (COR) gene expression. In addition, CBF-independent classes of BR-regulated COR genes are identified that are regulated in a BR- and CES-dependent manner during cold acclimation. A model is presented in which BRs govern different cold-responsive transcriptional cascades through the posttranslational modification of CES and redundantly acting factors. This contributes to the basal resistance against freezing stress, but also to the further improvement of this resistance through cold acclimation.

Improved nucleic acid extraction protocols for Ganoderma boninense, G. miniatocinctum and G. tornatum.

The first and most crucial step of all molecular techniques is to isolate high quality and intact nucleic acids. However, DNA and RNA isolation from fungal samples are usually difficult due to the cell walls that are relatively unsusceptible to lysis and often resistant to traditional extraction procedures. Although there are many extraction protocols for Ganoderma species, different extraction protocols have been applied to different species to obtain high yields of good quality nucleic acids, especially for genome and transcriptome sequencing. Ganoderma species, mainly G. boninense causes the basal stem rot disease, a devastating disease that plagues the oil palm industry. Here, we describe modified DNA extraction protocols for G. boninense, G. miniatocinctum and G. tornatum, and an RNA extraction protocol for G. boninense. The modified salting out DNA extraction protocol is suitable for G. boninense and G. miniatocinctum while the modified high salt and low pH protocol is suitable for G. tornatum. The modified DNA and RNA extraction protocols were able to produce high quality genomic DNA and total RNA of ~ 140 to 160 µg/g and ~ 80 µg/g of mycelia respectively, for Single Molecule Real Time (PacBio Sequel® System) and Illumina sequencing. These protocols will benefit those studying the oil palm pathogens at nucleotide level.

Adaptive resistance is not responsible for long-term drug resistance in a cellular model of triple negative breast cancer.

Resistance to cancer therapeutics represents a leading cause of mortality and is particularly important in cancers, such as triple negative breast cancer, for which no targeted therapy is available, as these are only treated with traditional chemotherapeutics. Cancer, as well as bacterial, drug resistance can be intrinsic, acquired or adaptive. Adaptive cancer drug resistance is gaining attention as a mechanism for the generation of long-term drug resistance as is the case with bacterial antibiotic resistance. We have used a cellular model of triple negative breast cancer (CAL51) and its drug resistance derivative (CALDOX) to gain insight into genome-wide expression changes associated with long-term doxorubicin (a widely used anthracycline for cancer treatment) resistance and doxorubicin-induced stress. Previous work indicates that both naïve and resistance cells have a functional p53-p21 axis controlling cell cycle at G1, although this is not a driver for drug resistance, but down-regulation of TOP2A (topoisomerase IIα). As expected, CALDOX cells have a signature characterized, in addition to down-regulation of TOP2A, by genes and pathways associated with drug resistance, metastasis and stemness. Both CAL51 and CALDOX stress signatures share 12 common genes (TRIM22, FAS, SPATA18, SULF2, CDKN1A, GDF15, MYO6, CXCL5, CROT, EPPK1, ZMAT3 and CD44), with roles in the above-mentioned pathways, indicating that these cells have similar functional responses to doxorubicin relaying on the p53 control of apoptosis. Eight genes are shared by both drug stress signatures (in CAL51 and CALDOX cells) and CALDOX resistant cells (FAS, SULF2, CDKN1A, CXCL5, CD44, SPATA18, TRIM22 and CROT), many of them targets of p53. This corroborates experimental data indicating that CALDOX cells, even in the absence of drug, have activated, at least partially, the p53-p21 axis and DNA damage response. Although this eight-gene signature might be an indicator of adaptive resistance, as this transient phenomenon due to short-term stress may not revert to its original state upon withdrawal of the stressor, previous experimental data indicates that the p53-p21 axis is not responsible for doxorubicin resistance. Importantly, TOP2A is not responsive to doxorubicin treatment and thus absent in both drug stress signatures. This indicates that during the generation of doxorubicin resistance, cells acquire genetic changes likely to be random, leading to down regulation of TOP2A, but selected during the generation of cells due to the presence of drug in the culture medium. This poses a considerable constraint for the development of strategies aimed at avoiding the emergence of drug resistance in the clinic.

Analysis of ripening-related gene expression in papaya using an Arabidopsis-based microarray.

BACKGROUND: Papaya (Carica papaya L.) is a commercially important crop that produces climacteric fruits with a soft and sweet pulp that contain a wide range of health promoting phytochemicals. Despite its importance, little is known about transcriptional modifications during papaya fruit ripening and their control. In this study we report the analysis of ripe papaya transcriptome by using a cross-species (XSpecies) microarray technique based on the phylogenetic proximity between papaya and Arabidopsis thaliana. RESULTS: Papaya transcriptome analyses resulted in the identification of 414 ripening-related genes with some having their expression validated by qPCR. The transcription profile was compared with that from ripening tomato and grape. There were many similarities between papaya and tomato especially with respect to the expression of genes encoding proteins involved in primary metabolism, regulation of transcription, biotic and abiotic stress and cell wall metabolism. XSpecies microarray data indicated that transcription factors (TFs) of the MADS-box, NAC and AP2/ERF gene families were involved in the control of papaya ripening and revealed that cell wall-related gene expression in papaya had similarities to the expression profiles seen in Arabidopsis during hypocotyl development. CONCLUSION: The cross-species array experiment identified a ripening-related set of genes in papaya allowing the comparison of transcription control between papaya and other fruit bearing taxa during the ripening process.

The Response of the Human Umbilical Vein Endothelial Cell Transcriptome to Variation in Magnesium Concentration.

Vascular endothelial cells have a critical role in the maintenance of cardiovascular function. Evidence suggests that endothelial function may be compromised under conditions of magnesium deficiency, which increases vulnerability to inflammation. Whole genome transcription analysis was used to explore the acute (24 h) effects of magnesium on human umbilical vascular endothelial cells (HUVEC) cultured in low (0.1 mM) or high (5 mM) concentrations. With low magnesium 2728 transcripts were differentially expressed compared to the 1 mM control cultures and 3030 were differentially expressed with high magnesium. 615 transcripts were differentially expressed under both conditions, of which only 34 showed a concentration-dependent response. Analysis indicated that cellular organisation and biogenesis and key cellular processes such as apoptosis were impacted by both low and high conditions. High magnesium also influenced protein binding functions, intracellular signal transduction, metabolic and catalytic processes. Both conditions impacted on stress-related processes, in particular the inflammatory response. Key mediators of calcium-dependent regulation of gene expression were responsive to both high and low magnesium conditions. The HUVEC transcriptome is highly sensitive to acute changes in the concentration of magnesium in culture medium. The findings of this study support the view that whilst inflammation is an important process that is responsive to magnesium, the function of the endothelium may be impacted by other magnesium-induced changes including maintenance of cellular integrity, receptor expression and metabolic functions. The high proportion of transcripts that did not show a concentration-dependent response suggests variation in magnesium may elicit indirect changes, possibly mediated by other ions.

Paternal low protein diet perturbs inter-generational metabolic homeostasis in a tissue-specific manner in mice.

The underlying mechanisms driving paternally-programmed metabolic disease in offspring remain poorly defined. We fed male C57BL/6 mice either a control normal protein diet (NPD; 18% protein) or an isocaloric low protein diet (LPD; 9% protein) for a minimum of 8 weeks. Using artificial insemination, in combination with vasectomised male mating, we generated offspring using either NPD or LPD sperm but in the presence of NPD or LPD seminal plasma. Offspring from either LPD sperm or seminal fluid display elevated body weight and tissue dyslipidaemia from just 3 weeks of age. These changes become more pronounced in adulthood, occurring in conjunction with altered hepatic metabolic and inflammatory pathway gene expression. Second generation offspring also display differential tissue lipid abundance, with profiles similar to those of first generation adults. These findings demonstrate that offspring metabolic homeostasis is perturbed in response to a suboptimal paternal diet with the effects still evident within a second generation.

Lipoprotein Deprivation Reveals a Cholesterol-Dependent Therapeutic Vulnerability in Diffuse Glioma Metabolism.

Poor outcomes associated with diffuse high-grade gliomas occur in both adults and children, despite substantial progress made in the molecular characterisation of the disease. Targeting the metabolic requirements of cancer cells represents an alternative therapeutic strategy to overcome the redundancy associated with cell signalling. Cholesterol is an integral component of cell membranes and is required by cancer cells to maintain growth and may also drive transformation. Here, we show that removal of exogenous cholesterol in the form of lipoproteins from culture medium was detrimental to the growth of two paediatric diffuse glioma cell lines, KNS42 and SF188, in association with S-phase elongation and a transcriptomic program, indicating dysregulated cholesterol homeostasis. Interrogation of metabolic perturbations under lipoprotein-deficient conditions revealed a reduced abundance of taurine-related metabolites and cholesterol ester species. Pharmacological reduction in intracellular cholesterol via decreased uptake and increased export was simulated using the liver X receptor agonist LXR-623, which reduced cellular viability in both adult and paediatric models of diffuse glioma, although the mechanism appeared to be cholesterol-independent in the latter. These results provide proof-of-principle for further assessment of liver X receptor agonists in paediatric diffuse glioma to complement the currently approved therapeutic regimens and expand the options available to clinicians to treat this highly debilitating disease.

Whole blood transcriptomic profiling identifies molecular pathways related to cardiovascular mortality in heart failure.

AIMS: Chronic heart failure (CHF) is a systemic syndrome with a poor prognosis and a need for novel therapies. We investigated whether whole blood transcriptomic profiling can provide new mechanistic insights into cardiovascular (CV) mortality in CHF. METHODS AND RESULTS: Transcriptome profiles were generated at baseline from 944 CHF patients from the BIOSTAT-CHF study, of whom 626 survived and 318 died from a CV cause during a follow-up of 21 months. Multivariable analysis, including adjustment for cell count, identified 1153 genes (6.5%) that were differentially expressed between those that survived or died and strongly related to a validated clinical risk score for adverse prognosis. The differentially expressed genes mainly belonged to five non-redundant pathways: adaptive immune response, proteasome-mediated ubiquitin-dependent protein catabolic process, T-cell co-stimulation, positive regulation of T-cell proliferation, and erythrocyte development. These five pathways were selectively related (RV coefficients >0.20) with seven circulating protein biomarkers of CV mortality (fibroblast growth factor 23, soluble ST2, adrenomedullin, hepcidin, pentraxin-3, WAP 4-disulfide core domain 2, and interleukin-6) revealing an intricate relationship between immune and iron homeostasis. The pattern of survival-associated gene expression matched with 29 perturbagen-induced transcriptome signatures in the iLINCS drug-repurposing database, identifying drugs, approved for other clinical indications, that were able to reverse in vitro the molecular changes associated with adverse prognosis in CHF. CONCLUSION: Systematic modelling of the whole blood protein-coding transcriptome defined molecular pathways that provide a link between clinical risk factors and adverse CV prognosis in CHF, identifying both established and new potential therapeutic targets.

MicroRNA-495/TGF-ß/FOXC1 axis regulates multidrug resistance in metaplastic breast cancer cells.

Triple-negative metaplastic breast carcinoma (MBC) poses a significant treatment challenge due to lack of targeted therapies and chemotherapy resistance. We isolated a novel MBC cell line, BAS, which showed a molecular and phenotypic profile different from the only other metaplastic cell model, HS578T cells. To gain insight behind chemotherapeutic resistance, we generated doxorubicin (HS-DOX, BAS-DOX) and paclitaxel (HS-TX, BAS-TX) resistant derivatives of both cell lines. Drug sensitivity assays indicated a truly multidrug resistant (MDR) phenotype. Both BAS-DOX and BAS-TX showed up-regulation of FOXC1 and its experimental down-regulation re-sensitized cells to doxorubicin and paclitaxel. Experimental modulation of FOXC1 expression in MCF-7 and MDA-MB-231 cells corroborated its role in MDR. Genome-wide expression analyses identified gene expression signatures characterized by up-regulation of TGFB2, which encodes cytokine TGF-ß2, in both BAS-DOX and BAS-TX cells. Pharmacological inhibition of the TGF-ß pathway with galunisertib led to down-regulation of FOXC1 and increase in drug sensitivity in both BAS-DOX and BAS-TX cells. MicroRNA (miR) expression analyses identified high endogenous miR-495-3p levels in BAS cells that were downregulated in both BAS MDR cells. Transient expression of miR-495-3p mimic in BAS-DOX and BAS-TX cells caused downregulation of TGFB2 and FOXC1 and re-sensitized cells to doxorubicin and paclitaxel, whereas miR-495-3p inhibition in BAS cells led to increase in resistance to both drugs and up-regulation of TGFB2 and FOXC1. Together, these data suggest interplay between miR-495-3p, TGF-ß2 and FOXC1 regulating MDR in MBC and open the exploration of novel therapeutic strategies.

CG7379 and ING1 suppress cancer cell invasion by maintaining cell-cell junction integrity.

Approximately 90% of cancer-related deaths can be attributed to a tumour's ability to spread. We have identified CG7379, the fly orthologue of human ING1, as a potent invasion suppressor. ING1 is a type II tumour suppressor with well-established roles in the transcriptional regulation of genes that control cell proliferation, response to DNA damage, oncogene-induced senescence and apoptosis. Recent work suggests a possible role for ING1 in cancer cell invasion and metastasis, but the molecular mechanism underlying this observation is lacking. Our results show that reduced expression of CG7379 promotes invasion in vivo in Drosophila, reduces the junctional localization of several adherens and septate junction components, and severely disrupts cell-cell junction architecture. Similarly, ING1 knockdown significantly enhances invasion in vitro and disrupts E-cadherin distribution at cell-cell junctions. A transcriptome analysis reveals that loss of ING1 affects the expression of several junctional and cytoskeletal modulators, confirming ING1 as an invasion suppressor and a key regulator of cell-cell junction integrity.

TWIST1 DNA methylation is a cell marker of airway and parenchymal lung fibroblasts that are differentially methylated in asthma.

BACKGROUND: Mesenchymal fibroblasts are ubiquitous cells that maintain the extracellular matrix of organs. Within the lung, airway and parenchymal fibroblasts are crucial for lung development and are altered with disease, but it has been difficult to understand their roles due to the lack of distinct molecular markers. We studied genome-wide DNA methylation and gene expression in airway and parenchymal lung fibroblasts from healthy and asthmatic donors, to identify a robust cell marker and to determine if these cells are molecularly distinct in asthma. RESULTS: Airway (N = 8) and parenchymal (N = 15) lung fibroblasts from healthy individuals differed in the expression of 158 genes, and DNA methylation of 3936 CpGs (Bonferroni adjusted p value < 0.05). Differential DNA methylation between cell types was associated with differential expression of 42 genes, but no single DNA methylation CpG feature (location, effect size, number) defined the interaction. Replication of gene expression and DNA methylation in a second cohort identified TWIST1 gene expression, DNA methylation and protein expression as a cell marker of airway and parenchymal lung fibroblasts, with DNA methylation having 100% predictive discriminatory power. DNA methylation was differentially altered in parenchymal (112 regions) and airway fibroblasts (17 regions) with asthmatic status, with no overlap between regions. CONCLUSIONS: Differential methylation of TWIST1 is a robust cell marker of airway and parenchymal lung fibroblasts. Airway and parenchymal fibroblast DNA methylation are differentially altered in individuals with asthma, and the role of both cell types should be considered in the pathogenesis of asthma.

A Genetic Analysis of Tumor Progression in Drosophila Identifies the Cohesin Complex as a Suppressor of Individual and Collective Cell Invasion.

Metastasis is the leading cause of death for patients with cancer. Consequently it is imperative that we improve our understanding of the molecular mechanisms that underlie progression of tumor growth toward malignancy. Advances in genome characterization technologies have been very successful in identifying commonly mutated or misregulated genes in a variety of human cancers. However, the difficulty in evaluating whether these candidates drive tumor progression remains a major challenge. Using the genetic amenability of Drosophila melanogaster we generated tumors with specific genotypes in the living animal and carried out a detailed systematic loss-of-function analysis to identify conserved genes that enhance or suppress epithelial tumor progression. This enabled the discovery of functional cooperative regulators of invasion and the establishment of a network of conserved invasion suppressors. This includes constituents of the cohesin complex, whose loss of function either promotes individual or collective cell invasion, depending on the severity of effect on cohesin complex function.

Heterologous oligonucleotide microarrays for transcriptomics in a non-model species; a proof-of-concept study of drought stress in Musa.

BACKGROUND: 'Systems-wide' approaches such as microarray RNA-profiling are ideally suited to the study of the complex overlapping responses of plants to biotic and abiotic stresses. However, commercial microarrays are only available for a limited number of plant species and development costs are so substantial as to be prohibitive for most research groups. Here we evaluate the use of cross-hybridisation to Affymetrix oligonucleotide GeneChip(R) microarrays to profile the response of the banana (Musa spp.) leaf transcriptome to drought stress using a genomic DNA (gDNA)-based probe-selection strategy to improve the efficiency of detection of differentially expressed Musa transcripts. RESULTS: Following cross-hybridisation of Musa gDNA to the Rice GeneChip(R) Genome Array, ~33,700 gene-specific probe-sets had a sufficiently high degree of homology to be retained for transcriptomic analyses. In a proof-of-concept approach, pooled RNA representing a single biological replicate of control and drought stressed leaves of the Musa cultivar 'Cachaco' were hybridised to the Affymetrix Rice Genome Array. A total of 2,910 Musa gene homologues with a >2-fold difference in expression levels were subsequently identified. These drought-responsive transcripts included many functional classes associated with plant biotic and abiotic stress responses, as well as a range of regulatory genes known to be involved in coordinating abiotic stress responses. This latter group included members of the ERF, DREB, MYB, bZIP and bHLH transcription factor families. Fifty-two of these drought-sensitive Musa transcripts were homologous to genes underlying QTLs for drought and cold tolerance in rice, including in 2 instances QTLs associated with a single underlying gene. The list of drought-responsive transcripts also included genes identified in publicly-available comparative transcriptomics experiments. CONCLUSION: Our results demonstrate that despite the general paucity of nucleotide sequence data in Musa and only distant phylogenetic relations to rice, gDNA probe-based cross-hybridisation to the Rice GeneChip(R) is a highly promising strategy to study complex biological responses and illustrates the potential of such strategies for gene discovery in non-model species.

On the causes of outliers in Affymetrix GeneChip data.

We describe various types of outliers seen in Affymetrix GeneChip data. We have been able to utilise the data in the Gene Expression Omnibus to screen GeneChips across a range of scales, from single probes, to spatially adjacent fractions of arrays, to whole arrays, to whole experiments. In this review we describe a number of causes for why some reported intensities might be misleading on GeneChips.

Low-intensity microwave irradiation does not substantially alter gene expression in late larval and adult Caenorhabditis elegans.

Reports that low-intensity microwave radiation induces heat-shock reporter gene expression in the nematode, Caenorhabditis elegans, have recently been reinterpreted as a subtle thermal effect caused by slight heating. This study used a microwave exposure system (1.0 GHz, 0.5 W power input; SAR 0.9-3 mW kg(-1) for 6-well plates) that minimises temperature differentials between sham and exposed conditions (< or =0.1 degrees C). Parallel measurement and simulation studies of SAR distribution within this exposure system are presented. We compared five Affymetrix gene arrays of pooled triplicate RNA populations from sham-exposed L4/adult worms against five gene arrays of pooled RNA from microwave-exposed worms (taken from the same source population in each run). No genes showed consistent expression changes across all five comparisons, and all expression changes appeared modest after normalisation (< or =40% up- or down-regulated). The number of statistically significant differences in gene expression (846) was less than the false-positive rate expected by chance (1131). We conclude that the pattern of gene expression in L4/adult C. elegans is substantially unaffected by low-intensity microwave radiation; the minor changes observed in this study could well be false positives. As a positive control, we compared RNA samples from N2 worms subjected to a mild heat-shock treatment (30 degrees C) against controls at 26 degrees C (two gene arrays per condition). As expected, heat-shock genes are strongly up-regulated at 30 degrees C, particularly an hsp-70 family member (C12C8.1) and hsp-16.2. Under these heat-shock conditions, we confirmed that an hsp-16.2::GFP transgene was strongly up-regulated, whereas two non-heat-inducible transgenes (daf-16::GFP; cyp-34A9::GFP) showed little change in expression.

Repurposing Antibacterial AM404 as a Potential Anticancer Drug for Targeting Colorectal Cancer Stem-Like Cells.

Tumour-promoting inflammation is involved in colorectal cancer (CRC) development and therapeutic resistance. However, the antibiotics and antibacterial drugs and signalling that regulate the potency of anticancer treatment upon forced differentiation of cancer stem-like cell (CSC) are not fully defined yet. We screened an NIH-clinical collection of the small-molecule compound library of antibacterial/anti-inflammatory agents that identified potential candidate drugs targeting CRC-SC for differentiation. Selected compounds were validated in both in vitro organoids and ex vivo colon explant models for their differentiation induction, impediment on neoplastic cell growth, and to elucidate the mechanism of their anticancer activity. We initially focused on AM404, an anandamide uptake inhibitor. AM404 is a metabolite of acetaminophen with antibacterial activity, which showed high potential in preventing CRC-SC features, such as stemness/de-differentiation, migration and drug-resistance. Furthermore, AM404 suppressed the expression of FBXL5 E3-ligase, where AM404 sensitivity was mimicked by FBXL5-knockout. This study uncovers a new molecular mechanism for AM404-altering FBXL5 oncogene which mediates chemo-resistance and CRC invasion, thereby proposes to repurpose antibacterial AM404 as an anticancer agent.

Metallome of cerebrovascular endothelial cells infected with Toxoplasma gondii using µ-XRF imaging and inductively coupled plasma mass spectrometry.

In this study, we measured the levels of elements in human brain microvascular endothelial cells (ECs) infected with T. gondii. ECs were infected with tachyzoites of the RH strain, and at 6, 24, and 48 hours post infection (hpi), the intracellular concentrations of elements were determined using a synchrotron-microfocus X-ray fluorescence microscopy (µ-XRF) system. This method enabled the quantification of the concentrations of Zn and Ca in infected and uninfected (control) ECs at sub-micron spatial resolution. T. gondii-hosting ECs contained less Zn than uninfected cells only at 48 hpi (p < 0.01). The level of Ca was not significantly different between infected and control cells (p > 0.05). Inductively Coupled Plasma Mass Spectrometry (ICP-MS) analysis revealed infection-specific metallome profiles characterized by significant increases in the intracellular levels of Zn, Fe, Mn and Cu at 48 hpi (p < 0.01), and significant reductions in the extracellular concentrations of Co, Cu, Mo, V, and Ag at 24 hpi (p < 0.05) compared with control cells. Zn constituted the largest part (74%) of the total metal composition (metallome) of the parasite. Gene expression analysis showed infection-specific upregulation in the expression of five genes, MT1JP, MT1M, MT1E, MT1F, and MT1X, belonging to the metallothionein gene family. These results point to a possible correlation between T. gondii infection and increased expression of MT1 isoforms and altered intracellular levels of elements, especially Zn and Fe. Taken together, a combined µ-XRF and ICP-MS approach is promising for studies of the role of elements in mediating host-parasite interaction.

Transcriptional analysis of adipose tissue during development reveals depot-specific responsiveness to maternal dietary supplementation.

Brown adipose tissue (BAT) undergoes pronounced changes after birth coincident with the loss of the BAT-specific uncoupling protein (UCP)1 and rapid fat growth. The extent to which this adaptation may vary between anatomical locations remains unknown, or whether the process is sensitive to maternal dietary supplementation. We, therefore, conducted a data mining based study on the major fat depots (i.e. epicardial, perirenal, sternal (which possess UCP1 at 7 days), subcutaneous and omental) (that do not possess UCP1) of young sheep during the first month of life. Initially we determined what effect adding 3% canola oil to the maternal diet has on mitochondrial protein abundance in those depots which possessed UCP1. This demonstrated that maternal dietary supplementation delayed the loss of mitochondrial proteins, with the amount of cytochrome C actually being increased. Using machine learning algorithms followed by weighted gene co-expression network analysis, we demonstrated that each depot could be segregated into a unique and concise set of modules containing co-expressed genes involved in adipose function. Finally using lipidomic analysis following the maternal dietary intervention, we confirmed the perirenal depot to be most responsive. These insights point at new research avenues for examining interventions to modulate fat development in early life.