Kinase Suppressor of RAS 1 (KSR1) Maintains the Transformed Phenotype of BRAFV600E Mutant Human Melanoma Cells.

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.

Clinicopathological Analysis of 907 Major and Minor Salivary Gland Tumors.

PURPOSE: The frequency and types of salivary gland tumors show significant geographical variations. The most common are primary epithelial tumors, with pleomorphic adenoma and mucoepidermoid carcinoma being the most frequent. This study aims to analyze the clinicopathological data of patients with major and minor salivary gland (MiSG) tumors. METHODS: The retrospective study included all patients with major and MiSG tumors diagnosed and treated between January 2000 and January 2019. Files of 907 patients were reviewed and investigated for clinicopathologic features of major and MiSG tumors in Serbia. RESULTS: The majority of tumors were of epithelial origin. Pleomorphic adenoma was the predominant type of tumor, with 35.1% among all tumors on all sites. Adenoid cystic carcinoma and mucoepider-moid carcinoma (with 7.1% and 2.7%, respectively) were the most common malignant ones. The most common localization was the parotid gland. Minor salivary gland tumors comprised 16.43% of all salivary gland tumors in our series, the most common localization being the oral cavity. The results of our study are mostly consistent with the results of other previously published studies. CONCLUSIONS: The most important finding, worth emphasizing, is that the most common malignant major and MiSG tumor in our population is adenoid cystic carcinoma, rather than mucoepidermoid carcinoma, in all investigated localizations. In addition, the nasal cavity is the most common localization among malignant MiSG tumors.

Calcium-pyro-hydrochar derived from the spent mushroom substrate as a functional sorbent of Pb2+ and Cd2+ from aqueous solutions.

A calcium-pyro-hydrochar (Ca-PHC) can be distinguished as a novel sorbent of Pb2+ and Cd2+ from an aqueous solution. It was obtained using hydrothermal treatment of the spent mushroom substrate (SMS), followed by a CaCl2·5H2O activation and pyrolysis. The characterisation of chars before and after modifications was done by scanning electron microscope (SEM), Brunauer-Emmett-Teller (BET) and Fourier transform infrared (FTIR). Batch experiments were performed to examine Ca-PHC's sorption properties and binding mechanisms to selected metal ions. The maximum sorption capacities of Ca-PHC for Pb2+ and Cd2+ were 297 mg g-1, and 131 mg g-1, respectively. The obtained results demonstrated that the sorption of Pb2+ and Cd2+ by Ca-PHC follows a pseudo-second kinetic model and Freundlich isotherm. The binding of the selected metals onto Ca-PHC was enabled by the ion-exchange mechanism, surface complexation, mineral precipitation and cation-π interaction. Thermodynamic parameters indicate that metal ions binding by Ca-PHC are spontaneous and endothermic. Due to the high adsorption capacities, the obtained Ca-PHC has good potential for application in industrial wastewater treatment. In addition, the demonstrated use of SMS highlights another possibility of applying this specific biomass relevant to sustainable and economical waste management in the growing mushroom industry.

The overexpression of SOX2 affects the migration of human teratocarcinoma cell line NT2/D1.

The altered expression of the SOX2 transcription factor is associated with oncogenic or tumor suppressor functions in human cancers. This factor regulates the migration and invasion of different cancer cells. In this study we investigated the effect of constitutive SOX2 overexpression on the migration and adhesion capacity of embryonal teratocarcinoma NT2/D1 cells derived from a metastasis of a human testicular germ cell tumor. We detected that increased SOX2 expression changed the speed, mode and path of cell migration, but not the adhesion ability of NT2/D1 cells. Additionally, we demonstrated that SOX2 overexpression increased the expression of the tumor suppressor protein p53 and the HDM2 oncogene. Our results contribute to the better understanding of the effect of SOX2 on the behavior of tumor cells originating from a human testicular germ cell tumor. Considering that NT2/D1 cells resemble cancer stem cells in many features, our results could contribute to the elucidation of the role of SOX2 in cancer stem cells behavior and the process of metastasis.

Late complication of the frontobasal trauma.

The injuries of the frontobasal region are always a great challenge to a surgeon, especially the management of bone defects of the frontal region. The authors present a patient with late, 33-year postaccident complication, which had been surgically treated, and whose frontal bone defect had been reconstructed with methyl methacrylate. Clinical examination and computed tomography confirmed signs of previous operation and presence of an infection and alloplastic material. Specific for this case was challenge to manage chronic infection and reestablish integrity of the skull in the frontal region. Out of a variety of autogenous or alloplastic materials, and considering the extent of bone defect and previous episodes of treatment aimed at aesthetic and functional results with good prognosis, we opted for reconstruction of the frontal region defect with combined titanium mesh impregnated with the hydroxyapatite cement.

Genomic and Transcriptomic Analysis of a Patient with Early-Onset Colorectal Cancer and Therapy-Induced Focal Nodular Hyperplasia: A Case Report.

Early-onset colorectal cancer (EOCRC), defined as colorectal cancer in individuals under 50 years of age, has shown an alarming increase in incidence worldwide. We report a case of a twenty-four-year-old female with a strong family history of colorectal cancer (CRC) but without an identified underlying genetic predisposition syndrome. Two years after primary surgery and adjuvant chemotherapy, the patient developed new liver lesions. Extensive diagnostic imaging was conducted to investigate suspected liver metastases, ultimately leading to a diagnosis of focal nodular hyperplasia. The young age of the patient has prompted comprehensive genomic and transcriptomic profiling in order to identify potential oncogenic drivers and inform further clinical management of the patient. Besides a number of oncogenic mutations identified in the patient's tumour sample, including KRAS G12D, TP53 R248W and TTN L28470V, we have also identified a homozygous deletion of 24.5 MB on chromosome 8. A multivariate Cox regression analysis of this patient's mutation profile conferred a favourable prognosis when compared with the TCGA COADREAD database. Notably, the identified deletion on chromosome 8 includes the WRN gene, which could contribute to the patient's overall positive response to chemotherapy. The complex clinical presentation, including the need for emergency surgery, early age at diagnosis, strong family history, and unexpected findings on surveillance imaging, necessitated a multidisciplinary approach involving medical, radiation, and surgical oncologists, along with psychological support and reproductive medicine specialists. Molecular profiling of the tumour strongly indicates that patients with complex mutational profile and rare genomic rearrangements require a prolonged surveillance and personalised informed interventions.

The role of modern imaging techniques in the diagnosis of malposition of the branch pulmonary arteries and possible association with microdeletion 22q11.2.

Malposition of the branch pulmonary arteries is a rare malformation with two forms. In the typical form, pulmonary arteries cross each other as they proceed to their respective lungs. The "lesser form" is characterised by the left pulmonary artery ostium lying directly superior to the ostium of the right pulmonary artery, without crossing of the branch pulmonary arteries. Malposition of the branch pulmonary arteries is often associated with other congenital heart defects and extracardiac anomalies, as well as with 22q11.2 microdeletion. We report three infants with crossed pulmonary arteries and one adolescent with "lesser form" of the malformation. The results suggest that diagnosis of malposition of the branch pulmonary arteries could be challenging if based solely on echocardiography, whereas modern imaging technologies such as contrast computed tomography and magnetic resonance angiography provide reliable establishment of diagnosis. In addition, we performed the first molecular characterisation of the 22q11.2 region among patients with malposition of the branch pulmonary arteries and revealed a 3-megabase deletion in two out of four patients

Retinoic acid receptor ß modulates mechanosensing and invasion in pancreatic cancer cells via myosin light chain 2.

Pancreatic ductal adenocarcinoma (PDAC) is the most common and lethal form of pancreatic cancer, characterised by stromal remodelling, elevated matrix stiffness and high metastatic rate. Retinoids, compounds derived from vitamin A, have a history of clinical use in cancer for their anti-proliferative and differentiation effects, and more recently have been explored as anti-stromal therapies in PDAC for their ability to induce mechanical quiescence in cancer associated fibroblasts. Here, we demonstrate that retinoic acid receptor ß (RAR-ß) transcriptionally represses myosin light chain 2 (MLC-2) expression in pancreatic cancer cells. As a key regulatory component of the contractile actomyosin machinery, MLC-2 downregulation results in decreased cytoskeletal stiffness and traction force generation, impaired response to mechanical stimuli via mechanosensing and reduced ability to invade through the basement membrane. This work highlights the potential of retinoids to target the mechanical drivers of pancreatic cancer.

Oncotherapeutic Strategies in Early Onset Colorectal Cancer.

Early onset colorectal cancer (EOCRC), defined as colorectal cancers in patients aged less than 50 years, is becoming an increasingly common issue, globally. Since 1994, the incidence of this condition has been rising by 2% annually. Approximately one in five patients under 50 years of age diagnosed with colorectal cancer have an underlying genetic predisposition syndrome. The detection of cancer among the other 80% of patients poses a considerable task, as there is no family history to advocate for commencing early screening in this group. Patients with EOCRC have distinct social, spiritual, fertility, and financial needs from their older counterparts that need to be addressed. This review discusses the risk factors associated with the development of EOCRC and current best practice for the management of this disease.

Transcriptional profiling of early differentiation of primary human mesenchymal stem cells into chondrocytes.

Articular cartilage has only very limited regenerative capacities in humans. Tissue engineering techniques for cartilage damage repair are limited in the production of hyaline cartilage. Mesenchymal stem/stromal cells (MSCs) are multipotent stem cells and can be differentiated into mature cartilage cells, chondrocytes, which could be used for repairing damaged cartilage. Chondrogenesis is a highly complex, relatively inefficient process lasting over 3 weeks in vitro. Methods: In order to better understand chondrogenic differentiation, especially the commitment phase, we have performed transcriptional profiling of MSC differentiation into chondrocytes from early timepoints starting 15 minutes after induction to 16 hours and fully differentiated chondrocytes at 21 days in triplicates.

Analysis of Alternative mRNA Splicing in Vemurafenib-Resistant Melanoma Cells.

Alternative mRNA splicing is common in cancers. In BRAF V600E-mutated malignant melanoma, a frequent mechanism of acquired resistance to BRAF inhibitors involves alternative splicing (AS) of BRAF. The resulting shortened BRAF protein constitutively dimerizes and conveys drug resistance. Here, we have analysed AS in SK-MEL-239 melanoma cells and a BRAF inhibitor (vemurafenib)-resistant derivative that expresses an AS, shortened BRAF V600E transcript. Transcriptome analysis showed differential expression of spliceosome components between the two cell lines. As there is no consensus approach to analysing AS events, we used and compared four common AS softwares based on different principles, DEXSeq, rMATS, ASpli, and LeafCutter. Two of them correctly identified the BRAF V600E AS in the vemurafenib-resistant cells. Only 12 AS events were identified by all four softwares. Testing the AS predictions experimentally showed that these overlapping predictions are highly accurate. Interestingly, they identified AS caused alterations in the expression of melanin synthesis and cell migration genes in the vemurafenib-resistant cells. This analysis shows that combining different AS analysis approaches produces reliable results and meaningful, biologically testable hypotheses.

Antioxidant Activity and Multi-Elemental Analysis of Dark Chocolate.

Cocoa beans are part of the cocoa plant fruit (Theobroma cacao L.) used to prepare various products such as chocolate, cocoa butter, jelly, liqueurs, cosmetics, etc. Dark chocolate is consumed worldwide by different populations and is known for its good taste, making it one of the most favoured food products. This work aimed to determine the content of total polyphenols (TPC), total flavonoids (TFC), and the antioxidant potential measured through the ability to scavenge DPPH free radicals (DPPH), ferric reducing power (FRAP), and total antioxidant capacity (TAC), as well as major and trace elements contained in twelve commercially available dark chocolate samples, with cocoa content ranging from 40% to 99%. The total polyphenols content ranged between 10.55 and 39.82 mg/g GAE, while the total flavonoid content was from 10.04 to 37.85 mg/g CE. All applied antioxidant assays indicate that the sample with the highest cocoa percentage shows the greatest antioxidant activity (DPPH: 48.34% of inhibition; FRAP: 89.00 mg/g GAE; TAC: 83.86 mg/g AAE). Statistical methods were applied to establish the differences between the samples concerning TPC, TFC, DPPH, FRAP and TAC, as well as to differentiate the samples according to the mineral content. The results indicated that the differences in TPC and TFC between different samples depended on the cocoa content and the addition of dried fruit pieces. A good correlation between antioxidant potency composite index (ACI) and declared cocoa content was noticed (R2 = 0.8034), indicating that the declared percentage of cocoa is a reliable indicator for antioxidant activity of analysed dark chocolate samples. The nutritional evaluation proved that the studied chocolate samples were an excellent source of Mg, Fe, Mn and Cu.

4q34.1-q35.2 deletion in a boy with phenotype resembling 22q11.2 deletion syndrome.

UNLABELLED: Small terminal or interstitial deletions involving bands 4q34 and 4q35 have been described in several patients with a relatively mild phenotype such as mild to moderate intellectual disability and minor dysmorphic features. We present a boy born from unrelated parents with a de novo 4q34.1-q35.2 deletion and clinical features resembling 22q11.2 deletion syndrome. To the best of our knowledge, this is the first reported patient with 4q34-q35 deletion and phenotype resembling 22q11.2 deletion syndrome without fifth finger anomalies as a specific feature of 4q- syndrome. G-banding karyotyping disclosed the deletion, which was further delineated by microarray comparative genomic hybridization. Fluorescence in situ hybridization and multiplex ligation-dependent probe amplification analyses did not reveal rearrangements of 22q11.2 region. MLPA confirmed the deletion within the 4q35.2 region. CONCLUSION: Given the considerable clinical overlaps between the 22q11.2 deletion syndrome and clinical manifestation of the patient described in this study, we propose that region 4q34.1-q35.2 should be considered as another region associated with phenotype resembling 22q11.2 deletion syndrome. We also propose that distal 4q deletions should be considered in the evaluation of patients with phenotypic manifestations resembling 22q11.2 deletion syndrome in whom no 22q11.2 microdeletion was detected, even in the absence of distinctive fifth finger anomalies. Additionally, we underline the importance of applying array CGH that enables simultaneous genome-wide detection and delineation of copy number changes (e.g., deletions and duplications).

A Chemo-Genomic Approach Identifies Diverse Epigenetic Therapeutic Vulnerabilities in MYCN-Amplified Neuroblastoma.

Although a rare disease, neuroblastoma accounts for the highest proportion of childhood cancer deaths. There is a lack of recurrent somatic mutations in neuroblastoma embryonal tumours, suggesting a possible role for epigenetic alterations in driving this cancer. While an increasing number of reports suggest an association of MYCN with epigenetic machinery, the mechanisms of these interactions are poorly understood in the neuroblastoma setting. Utilising chemo-genomic approaches we revealed global MYCN-epigenetic interactions and identified numerous epigenetic proteins as MYCN targets. The epigenetic regulators HDAC2, CBX8 and CBP (CREBBP) were all MYCN target genes and also putative MYCN interactors. MYCN-related epigenetic genes included SMARCs, HDACs, SMYDs, BRDs and CREBBP. Expression levels of the majority of MYCN-related epigenetic genes showed predictive ability for neuroblastoma patient outcome. Furthermore, a compound library screen targeting epigenetic proteins revealed broad susceptibility of neuroblastoma cells to all classes of epigenetic regulators, belonging to families of bromodomains, HDACs, HATs, histone methyltransferases, DNA methyltransferases and lysin demethylases. Ninety-six percent of the compounds reduced MYCN-amplified neuroblastoma cell viability. We show that the C646 (CBP-bromodomain targeting compound) exhibits switch-like temporal and dose response behaviour and is effective at reducing neuroblastoma viability. Responsiveness correlates with MYCN expression, with MYCN-amplified cells being more susceptible to C646 treatment. Thus, exploiting the broad vulnerability of neuroblastoma cells to epigenetic targeting compounds represents an exciting strategy in neuroblastoma treatment, particularly for high-risk MYCN-amplified tumours.

Molecular characterization of a marine turtle tumor epizootic, profiling external, internal and postsurgical regrowth tumors.

Sea turtle populations are under threat from an epizootic tumor disease (animal epidemic) known as fibropapillomatosis. Fibropapillomatosis continues to spread geographically, with prevalence of the disease also growing at many longer-affected sites globally. However, we do not yet understand the precise environmental, mutational and viral events driving fibropapillomatosis tumor formation and progression.Here we perform transcriptomic and immunohistochemical profiling of five fibropapillomatosis tumor types: external new, established and postsurgical regrowth tumors, and internal lung and kidney tumors. We reveal that internal tumors are molecularly distinct from the more common external tumors. However, they have a small number of conserved potentially therapeutically targetable molecular vulnerabilities in common, such as the MAPK, Wnt, TGFß and TNF oncogenic signaling pathways. These conserved oncogenic drivers recapitulate remarkably well the core pan-cancer drivers responsible for human cancers. Fibropapillomatosis has been considered benign, but metastatic-related transcriptional signatures are strongly activated in kidney and established external tumors. Tumors in turtles with poor outcomes (died/euthanized) have genes associated with apoptosis and immune function suppressed, with these genes providing putative predictive biomarkers.Together, these results offer an improved understanding of fibropapillomatosis tumorigenesis and provide insights into the origins, inter-tumor relationships, and therapeutic treatment for this wildlife epizootic.

Comparative analysis of SOX3 protein orthologs: Expansion of homopolymeric amino acid tracts during vertebrate evolution.

To understand more fully the structure and evolution of the SOX3 protein, we comparatively analyzed its orthologs in vertebrates. Since complex disorders are associated with human SOX3 polyalanine expansions, our investigation focused on both compositional and evolutionary analysis of various homopolymeric amino acid tracts observed in SOX3 orthologs. Our analysis revealed that the observed homopolymeric alanine, glycine, and proline tracts are mammal-specific, except for one polyglycine tract present in birds. Since it is likely that the SOX3 protein acquired additional roles in brain development in Eutheria, we might speculate that development of novel brain functions during the course of evolution was affected, at least in part, by such structural-functional changes in the SOX3 protein.

Extensive rewiring of the EGFR network in colorectal cancer cells expressing transforming levels of KRASG13D.

Protein-protein-interaction networks (PPINs) organize fundamental biological processes, but how oncogenic mutations impact these interactions and their functions at a network-level scale is poorly understood. Here, we analyze how a common oncogenic KRAS mutation (KRASG13D) affects PPIN structure and function of the Epidermal Growth Factor Receptor (EGFR) network in colorectal cancer (CRC) cells. Mapping >6000 PPIs shows that this network is extensively rewired in cells expressing transforming levels of KRASG13D (mtKRAS). The factors driving PPIN rewiring are multifactorial including changes in protein expression and phosphorylation. Mathematical modelling also suggests that the binding dynamics of low and high affinity KRAS interactors contribute to rewiring. PPIN rewiring substantially alters the composition of protein complexes, signal flow, transcriptional regulation, and cellular phenotype. These changes are validated by targeted and global experimental analysis. Importantly, genetic alterations in the most extensively rewired PPIN nodes occur frequently in CRC and are prognostic of poor patient outcomes.

Prenatal diagnosis of Pallister-Killian syndrome in young woman: ultrasound indicators and confirmation by FISH.

We report the first case of Pallister-Killian syndrome diagnosed prenatally in Western Balkan region where one of the ultrasound markers was intrauterine growth restriction. During routine ultrasound control of the pregnancy at 21st gestation week (second pregnancy of the 25 year old woman) symmetrical intrauterine growth restriction (IUGR), short long bones, ventriculomegaly and oligoamnion were noted. Amniotic fluid was examined cytogenetically. Fetal karyotype obtained by GTG banding of amniocytes revealed mosaic female karyotype 46,XX/47,XX,+mar (F-like). C-banding indicated that F-like marker does not belong to F, E or G chromosomal group. Employing targeted FISH with arm-specific probe for chromosome 12, tetrasomy 12p was confirmed. Fetal lymphocytes revealed normal female karyotype. This case showed that i(12p) could be found in pregnancy of young woman, not only in those of advanced age, as usually reported in the literature. This case also showed that intrauterine growth restriction could be one of the ultrasound markers associated with Pallister-Killian syndrome.

An Integrative Computational Approach for a Prioritization of Key Transcription Regulators Associated With Nanomaterial-Induced Toxicity.

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.

PHD3 Regulates p53 Protein Stability by Hydroxylating Proline 359.

Cellular p53 protein levels are regulated by a ubiquitination/de-ubiquitination cycle that can target the protein for proteasomal destruction. The ubiquitination reaction is catalyzed by a multitude of ligases, whereas the removal of ubiquitin chains is mediated by two deubiquitinating enzymes (DUBs), USP7 (HAUSP) and USP10. Here, we show that PHD3 hydroxylates p53 at proline 359, a residue that is in the p53-DUB binding domain. Hydroxylation of p53 upon proline 359 regulates its interaction with USP7 and USP10, and its inhibition decreases the association of p53 with USP7/USP10, increases p53 ubiquitination, and rapidly reduces p53 protein levels independently of mRNA expression. Our results show that p53 is a PHD3 substrate and that hydroxylation by PHD3 regulates p53 protein stability through modulation of ubiquitination.