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Kinase Suppressor of RAS 1 (KSR1) is a scaffolding protein for the RAS-RAF-MEK-ERK pathway, which is one of the most frequently altered pathways in human cancers. Previous results have shown that KSR1 has a critical role in mutant RAS-mediated transformation. Here, we examined the role of KSR1 in mutant BRAF transformation. We used CRISPR/Cas9 to knock out KSR1 in a BRAFV600E-transformed melanoma cell line. KSR1 loss produced a complex phenotype characterised by impaired proliferation, cell cycle defects, decreased transformation, decreased invasive migration, increased cellular senescence, and increased apoptosis. To decipher this phenotype, we used a combination of proteomic ERK substrate profiling, global protein expression profiling, and biochemical validation assays. The results suggest that KSR1 directs ERK to phosphorylate substrates that have a critical role in ensuring cell survival. The results further indicate that KSR1 loss induces the activation of p38 Mitogen-Activated Protein Kinase (MAPK) and subsequent cell cycle aberrations and senescence. In summary, KSR1 function plays a key role in oncogenic BRAF transformation. [ABSTRACT FROM AUTHOR]

Inhalation of multi-walled carbon nanotubes (MWCNTs) induces lung inflammation. Depending on industrial applications, CNTs with different physicochemical characteristics are produced and workers can potentially be exposed. This raises concerns about the long-term health effects of these nanomaterials. Because of the wide variety of MWCNTs, it is essential to study the toxicological effects of CNTs of various shapes and to better understand the impact physical and chemical properties have on their toxicity. In this study, rats were exposed by nose-only to two pristine MWCNTs with different morphologies: the long and thick NM-401 or the short and thin NM-403. After four weeks of inhalation, animals were euthanized at four different times during the recovery period: three days (short-term), 30 and 90 days (intermediate-term) and 180 days (long-term). Analyses of the transcriptome in the whole lung and the proteome in the bronchoalveolar lavage fluid of exposed animals were performed to understand the MWCNT underlying mechanisms of toxicity. Following inhalation of NM-401, we observed a dose-dependent increase in the number of differentially expressed genes and proteins, whereas there is no clear difference between the two concentrations of NM-403. After NM-403 inhalation, the number of differentially expressed genes and proteins varied less between the four post-exposure times compared to NM-401, which supports the postulation of a persistent effect of this type of CNT. Our toxicogenomics approaches give insights into the different toxicological profile following MWCNT exposure. [ABSTRACT FROM AUTHOR]

Rationale: Mutations in the cystic fibrosis transmembrane regulator (CFTR) gene form the basis of cystic fibrosis (CF). There remains an important knowledge gap in CF as to how diminished CFTR activity leads to the dominant inflammatory response within CF airways.Objectives: To investigate if extracellular vesicles (EVs) contribute to inflammatory signalling in CF.Methods: EVs released from CFBE41o-, CuFi-5, 16HBE14o- and NuLi-1 cells were characterised by nanoparticle tracking analysis (NTA). EVs isolated from bronchoalveolar lavage fluid (BALF) from 30 people with CF (PWCF) were analysed by NTA and mass spectrometry and compared with controls. Neutrophils were isolated from the blood of 8 PWCF to examine neutrophil migration in the presence of CFBE41o- EVs.Results: A significantly higher level of EVs were released from CFBE41o- (p<0.0001) and CuFi-5 (p=0.0209) relative to control cell lines. A significantly higher level of EVs were detected in BALF of PWCF, in three different age groups relative to controls (p=0.01, 0.001, 0.002). A significantly lower level of EVs were released from CFBE41o- (p<0.001) and CuFi-5 (p=0.0002) cell lines treated with CFTR modulators. Significant changes in the protein expression of 126 unique proteins was determined in EVs obtained from the BALF of PWCF of different age groups (p<0.001-0.05). A significant increase in chemotaxis of neutrophils derived from PWCF was observed in the presence of CFBE41o EVs (p=0.0024) compared with controls.Conclusion: This study demonstrates that EVs are produced in CF airway cells, have differential protein expression at different ages and drive neutrophil recruitment in CF. [ABSTRACT FROM AUTHOR]

A rapid increase of new nanomaterial (NM) products poses new challenges for their risk assessment. Current traditional methods for estimating potential adverse health effect of NMs are complex, time consuming, and expensive. In order to develop new prediction tests for nanotoxicity evaluation, a systems biology approach, and data from high-throughput omics experiments can be used. We present a computational approach that combines reverse engineering techniques, network analysis and pathway enrichment analysis for inferring the transcriptional regulation landscape and its functional interpretation. To illustrate this approach, we used published transcriptomic data derived from mice lung tissue exposed to carbon nanotubes (NM-401 and NRCWE-26). Because fibrosis is the most common adverse effect of these NMs, we included in our analysis the data for bleomycin (BLM) treatment, which is a well-known fibrosis inducer. We inferred gene regulatory networks for each NM and BLM to capture functional hierarchical regulatory structures between genes and their regulators. Despite the different nature of the lung injury caused by nanoparticles and BLM, we identified several conserved core regulators for all agents. We reason that these regulators can be considered as early predictors of toxic responses after NMs exposure. This integrative approach, which refines traditional methods of transcriptomic analysis, can be useful for prioritization of potential core regulators and generation of new hypothesis about mechanisms of nanoparticles toxicity. [ABSTRACT FROM AUTHOR]

Rationale: Obesity is a risk factor for atherothrombosis and various cancers. However, the mechanisms are not yet completely clarified. Objectives: We aimed to verify whether the microparticles (MPs) released from thrombin-activated platelets differed in obese and non-obese women for number, size, and proteomics cargo and the capacity to modulate in vitro the expression of (i) genes related to the epithelial to mesenchymal transition (EMT) and the endothelial to mesenchymal transition (EndMT), and (ii) cyclooxygenase (COX)-2 involved in the production of angiogenic and inflammatory mediators. Methods and Results: MPs were obtained from thrombin activated platelets of four obese and their matched non-obese women. MPs were analyzed by cytofluorimeter and protein content by liquid chromatography-mass spectrometry. MPs from obese women were not different in number but showed increased heterogeneity in size. In obese individuals, MPs containing mitochondria (mitoMPs) expressed lower CD41 levels and increased phosphatidylserine associated with enhanced Factor V representing a signature of a prothrombotic state. Proteomics analysis identified 44 proteins downregulated and three upregulated in MPs obtained from obese vs. non-obese women. A reduction in the proteins of the α-granular membrane and those involved in mitophagy and antioxidant defenses-granular membrane was detected in the MPs of obese individuals. MPs released from platelets of obese individuals were more prone to induce the expression of marker genes of EMT and EndMT when incubated with human colorectal cancer cells (HT29) and human cardiac microvascular endothelial cells (HCMEC), respectively. A protein, highly enhanced in obese MPs, was the pro-platelet basic protein with pro-inflammatory and tumorigenic actions. Exclusively MPs from obese women induced COX-2 in HCMEC. Conclusion: Platelet-derived MPs of obese women showed higher heterogeneity in size and contained different levels of proteins relevant to thrombosis and tumorigenesis. MPs from obese individuals presented enhanced capacity to cause changes in the expression of EMT and EndMT marker genes and to induce COX-2. These effects might contribute to the increased risk for the development of thrombosis and multiple malignancies in obesity. Clinical Trial Registration: www.ClinicalTrials.gov, identifier NCT01581801. [ABSTRACT FROM AUTHOR]

This paper focuses on the role that mitogen-activated protein kinases (MAPKs) play in drug-induced kidney injury. The MAPKs, of which there are four major classes (ERK, p38, JNK, and ERK5/BMK), are signalling cascades which have been found to be broadly conserved across a wide variety of organisms. MAPKs allow effective transmission of information from the cell surface to the cytosolic or nuclear compartments. Cross talk between the MAPKs themselves and with other signalling pathways allows the cell to modulate responses to a wide variety of external stimuli. The MAPKs have been shown to play key roles in both mediating and ameliorating cellular responses to stress including xenobiotic-induced toxicity. Therefore, this paper will discuss the specific role of the MAPKs in the kidney in response to injury by a variety of xenobiotics and the potential for therapeutic intervention at the level of MAPK signalling across different types of kidney disease. [ABSTRACT FROM AUTHOR]

The natural compound curcumin has been shown to have therapeutic potential against a wide range of diseases such as cancer. Curcumin reduces cell viability of renal cell carcinoma (RCC) cells when combined with TNF-related apoptosis-inducing ligand (TRAIL), a cytokine that specifically targets cancer cells, by helping overcome TRAIL resistance. However, the therapeutic effects of curcumin are limited by its low bioavailability. Similar compounds to curcumin with higher bioavailability, such as demethoxycurcumin (DMC) and 3,5-bis(2-fluorobenzylidene)-4-piperidone (EF24), can potentially have similar anticancer effects and show a similar synergy with TRAIL, thus reducing RCC viability. This study aims to show the effects of DMC and EF24 in combination with TRAIL at reducing ACHN cell viability and ACHN cell migration. It also shows the changes in death receptor 4 (DR4) expression after treatment with these compounds individually and in combination with TRAIL, which can play a role in their mechanism of action. [ABSTRACT FROM AUTHOR]

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), is a selective anticancer cytokine capable of exerting a targeted therapy approach. Disappointingly, recent research has highlighted the development of TRAIL resistance in cancer cells, thus minimising its usefulness in clinical settings. However, several recent studies have demonstrated that cancer cells can be sensitised to TRAIL through the employment of a combinatorial approach, utilizing TRAIL in conjunction with other natural or synthetic anticancer agents. In the present study, the chemo-sensitising effect of curcumin on TRAIL-induced apoptosis in renal carcinoma cells (RCC) was investigated. The results indicate that exposure of kidney cancer ACHN cells to curcumin sensitised the cells to TRAIL, with the combination treatment of TRAIL and curcumin synergistically targeting the cancer cells without affecting the normal renal proximal tubular epithelial cells (RPTEC/TERT1) cells. Furthermore, this combination treatment was shown to induce caspase-dependent apoptosis, inhibition of the proteasome, induction of ROS, upregulation of death receptor 4 (DR4), alterations in mitogen-activated protein kinase (MAPK) signalling and induction of endoplasmic reticulum stress. An in vivo zebrafish embryo study demonstrated the effectiveness of the combinatorial regime to inhibit tumour formation without affecting zebrafish embryo viability or development. Overall, the results arising from this study demonstrate that curcumin has the ability to sensitise TRAIL-resistant ACHN cells to TRAIL-induced apoptosis. [ABSTRACT FROM AUTHOR]

Patients with neuropathic pain have altered proteomic and neuropeptide constituents in cerebrospinal fluid (CSF) compared to controls. Tonic spinal cord stimulation (SCS) has demonstrated differential expression of neuropeptides in CSF before and after treatment suggesting potential mechanisms of action. Burst-SCS is an evidence-based paraesthesia free waveform utilised for neuropathic pain with a potentially different mechanistic action to tonic SCS. This study examines the dynamic biological changes of CSF at a cellular and proteome level after Burst-SCS. Patients with neuropathic pain selected for SCS had CSF sampled prior to implant of SCS and following 8 weeks of continuous Burst-SCS. Baseline and 8-week pain scores with demographics were recorded. T cell frequencies were analysed by flow cytometry, proteome analysis was performed using mass spectrometry and secreted cytokines, chemokines and neurotrophins were measured by enzyme-linked immunosorbent assay (ELISA). 4 patients (2 females, 2 males) with a mean age of 51 years (+/-SEM 2.74, SD 5.48) achieved a reduction in pain of >50% following 8 weeks of Burst-SCS. Analysis of the CSF proteome indicated a significant alteration in protein expression most related to synapse assembly and immune regulators. There was significantly lower expression of the proteins: growth hormone A1 (PRL), somatostatin (SST), nucleobindin-2 (NUCB2), Calbindin (CALB1), acyl-CoA binding protein (DBI), proSAAS (PCSK1N), endothelin-3 (END3) and cholecystokinin (CCK) after Burst-SCS. The concentrations of secreted chemokines and cytokines and the frequencies of T cells were not significantly changed following Burst-SCS. This study characterised the alteration in the CSF proteome in response to burst SCS in vivo. Functional analysis indicated that the alterations in the CSF proteome is predominately linked to synapse assembly and immune effectors. Individual protein analysis also suggests potential supraspinal mechanisms. Unlabelled Image • Burst stimulation for chronic pain modulates the proteome of spinal fluid in vivo. • Proteins in synapse assembly are altered suggesting a potential mechanism of action. • Immune effectors were the most upregulated proteins after Burst stimulation. • PRL, SST and NUCB2 showed decreased expression suggesting supraspinal effects. [ABSTRACT FROM AUTHOR]