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Supplementary Figures 1-3 from Genetic Unmasking of an Epigenetically Silenced microRNA in Human Cancer Cells

Supplementary Table 1 from Genetic Unmasking of an Epigenetically Silenced microRNA in Human Cancer Cells

The mechanisms underlying microRNA (miRNA) disruption in human disease are poorly understood. In cancer cells, the transcriptional silencing of tumor suppressor genes by CpG island promoter hypermethylation has emerged as a common hallmark. We wondered if the same epigenetic disruption can “hit” miRNAs in transformed cells. To address this issue, we have used cancer cells genetically deficient for the DNA methyltransferase enzymes in combination with a miRNA expression profiling. We have observed that DNA hypomethylation induces a release of miRNA silencing in cancer cells. One of the main targets is miRNA-124a, which undergoes transcriptional inactivation by CpG island hypermethylation in human tumors from different cell types. Interestingly, we functionally link the epigenetic loss of miRNA-124a with the activation of cyclin D kinase 6, a bona fide oncogenic factor, and the phosphorylation of the retinoblastoma, a tumor suppressor gene. [Cancer Res 2007;67(4):1424–9]

At the end of the nineteenth century, massive population declines were observed in carnivores due to the emergence of infectious diseases. This study aims to investigate, by means of coprological analysis, the prevalence and intensity of the parasites that infect the endangered Iberian wolf Canis lupus signatus and two mesocarnivores (the red fox Vulpes vulpes and the stone marten Martes foina) in Central Portugal. In total, 67.2% of the samples screened were infected; Toxascaris leonina (40.6%) was the parasite with the highest prevalence, followed by Ancylostomatidae and Eimeria spp. (28.1%). Eimeria spp. was found in stone marten with the highest infection rate (37,800 OPG), followed by T. leonina (10,100 EPG) in a red fox sample. Moderate to high levels of parasitic infections were identified in 73.3% of red foxes from the western area. Our results highlight the possibility of cross-infection among these carnivore species and cross-contamination in the wildlife-livestock-human interface.

This work presents three data-driven models based on process data, to estimate different indicators related to process performance in a steel production process. The generated models allow the optimization of the process parameters to achieve optimal performance and quality levels. A new approach based on ensembles has been developed with feature selection methods and four state-of-the-art regression approximations (random forest, gradient boosting, xgboost and neural networks). The results show that the proposed approach makes the prediction more stable reducing the variance for all cases, even in one case, slightly reducing the bias. Furthermore, from the four machine learning paradigms presented, random forest is the one with the best results in a quantitative way, obtaining a coefficient of determination of 0.98 as a maximum, depending on the target sub-process. This research is supported by the European Union’s Horizon 2020 Research and Innovation Framework Programme [grant agreement No 723661; COCOP; http://www.cocop-spire.eu (accessed on 6 January 2022)]. The authors want to acknowledge the work of the whole COCOP consortium.

The FixK protein plays a pivotal role in a complex regulatory network, which controls genes for microoxic, denitrifying, and symbiotic nitrogen-fixing lifestyles in Bradyrhizobium diazoefficiens. Among the microoxic-responsive FixK-activated genes are the fixNOQP operon, indispensable for respiration in symbiosis, and the nnrR regulatory gene needed for the nitric-oxide dependent induction of the norCBQD genes encoding the denitrifying nitric oxide reductase. FixK is a CRP/FNR-type transcription factor, which recognizes a 14 bp-palindrome (FixK box) at the regulated promoters through three residues (L195, E196, and R200) within a C-terminal helix-turn-helix motif. Here, we mapped the determinants for discriminatory FixK-mediated regulation. While R200 was essential for DNA binding and activity of FixK, L195 was involved in protein–DNA complex stability. Mutation at positions 1, 3, or 11 in the genuine FixK box at the fixNOQP promoter impaired transcription activation by FixK, which was residual when a second mutation affecting the box palindromy was introduced. The substitution of nucleotide 11 within the NnrR box at the norCBQD promoter allowed FixK-mediated activation in response to microoxia. Thus, position 11 within the FixK/NnrR boxes constitutes a key element that changes FixK targets specificity, and consequently, it might modulate B. diazoefficiens lifestyle as nitrogen fixer or as denitrifier., This work was funded by Fondo Europeo de Desarrollo Regional (FEDER)-co-financed grants AGL2011-23383 and AGL2015-63651-P to S.M., and AGL2013-45087-R and AGL2017-85676-R to M.J.D. [Ministerio de Economía y Competitividad, presently Ministerio de Ciencia e Innovación (MICINN), Spain]. Grants P12-AGR-1968 to E.J.B., and P18-RT-1401 to S.M. and M.J.D., and support from the Junta de Andalucía, Spain, to Group BIO-275 are also acknowledged. J.J.C., A.J.-L., and M.J.T. were supported by contracts funded by grants AGL2015-63651-P, AGL2017-85676-R, and AGL2013-45087-R, respectively. J.J.C. also acknowledged to Alfonso Martín Escudero Foundation the support by its abroad postdoctoral grants program. A.J.-L. was also financed by grant P12-AGR-1968 during her PhD thesis' period. S.P. acknowledged the FPU Program (Ministerio de Educación, Cultura y Deporte, presently Ministerio de Universidades, Spain; grant FPU2015/04716) for financial support. Juan Sanjuán (Estación Experimental de Zaidín, CSIC, Granada, Spain) is acknowledged for his critical comments on the manuscript. We are grateful to the financial support of the publication fee by the CSIC Open Access Publication Support Program through its Unit of Scientific Information Resources for Research (URICI). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

PD was supported by a grant of the Netherlands Scientific Organization NWO 824.15.023 to RPdV. The Academy of Finland grant no. 308284 to MRM is acknowledged. Part of the research was performed at the Environmental Molecular Sciences Laboratory (EMSL), a national scientific user facility sponsored by the Department of Energy's Office of Biological and Environmental Research, located at the Pacific Northwest National Laboratory in Richland, WA, USA. The work conducted by the U.S. Department of Energy Joint Genome Institute (JGI), was supported by the Office of Science of the U.S. Department of Energy under Contract No. DE‐AC02‐05CH11231. CSL was supported by the CatchBio program. White‐rot fungi, such as Dichomitus squalens, degrade all wood components and inhabit mixed‐wood forests containing both soft‐ and hardwood species. In this study, we evaluated how D. squalens responded to the compositional differences in softwood [guaiacyl (G) lignin and higher mannan content] and hardwood [syringyl/guaiacyl (S/G) lignin and higher xylan content] using semi‐natural solid cultures. Spruce (softwood) and birch (hardwood) sticks were degraded by D. squalens as measured by oxidation of the lignins using 2D‐NMR. The fungal response as measured by transcriptomics, proteomics and enzyme activities showed a partial tailoring to wood composition. Mannanolytic transcripts and proteins were more abundant in spruce cultures, while a proportionally higher xylanolytic activity was detected in birch cultures. Both wood types induced manganese peroxidases to a much higher level than laccases, but higher transcript and protein levels of the manganese peroxidases were observed on the G‐lignin rich spruce. Overall, the molecular responses demonstrated a stronger adaptation to the spruce rather than birch composition, possibly because D. squalens is mainly found degrading softwoods in nature, which supports the ability of the solid wood cultures to reflect the natural environment. Publisher PDF

Dichomitus squalens is a white-rot fungus that colonizes and grows mainly on softwood and is commonly found in the northern parts of Europe, North America, and Asia. We analyzed the genetic and physiological diversity of eight D. squalens monokaryons derived from a single dikaryon. In addition, an unrelated dikaryon and a newly established dikaryon from two of the studied monokaryons were included. Both growth and lignocellulose acting enzyme profiles were highly variable between the studied monokaryotic and dikaryotic strains, demonstrating a high level of diversity within the species.

Author(s): Daly, Paul; Peng, Mao; Casado Lopez, Sara; Lipzen, Anna; Ng, Vivian; Singan, Vasanth R; Wang, Mei; Grigoriev, Igor V; de Vries, Ronald P; Makela, Miia R | Abstract: In Section 3.1 of the Results and Discussion, it should have stated 100 μM and not 50 μM for the concentration of the aromatic compounds. The correct concentration was stated in the Materials and Methods and the error only occurred in the Results and Discussion section. The authors would like to apologize for any inconvenience caused.

Parkinson's disease has been widely related to both apoptosis and oxidative stress. Many publications relate the loss of mitochondrial potential to an apoptosis-mediated cell death in different in vivo and in vitro models of this pathology. The present study used the dopaminegic specific neurotoxin 1-methyl-4-phenylpyridinium (MPP(+) ) on neuron-like PC12 cells, which is a well-accepted model of Parkinson's disease. Results showed an early increase in oxidants, which drives the modulation of c-Jun N-terminal kinase (JNK) and AKT/mammalian target of rapamycin (mTOR) pathways, mimicking peroxide treatment. However, the cell death found in neuronal PC12 cells treated with MPP(+) was not a caspase-associated apoptosis. Electron microscopic images illustrated autophagic cell death, which was confirmed by a Beclin-1 and ATG expression increase, accumulation of acidic vesicles, and rescue by an autophagy inhibitor. In conclusion, the boost in oxidants from MPP(+) treatment in neuronal PC12 is modulating both survival (AKT/mTOR) and death (JNK) pathways, which are the perpetrators of an autophagic cell death.

Background: Despite recent advances in cancer therapy, the 5-year survival rate for Ewing's sarcoma is still very low, and new therapeutic approaches are necessary. It was found previously that melatonin induces cell death in the Ewing's sarcoma cell line, SK-N-MC, by activating the extrinsic apoptotic pathway. Methods: Melatonin actions were analysed by metabolic viability/survival cell assays, flow cytometry, quantitative PCR for mRNA expression, western blot for protein activation/expression and electrophoretic mobility shift assay for transcription factor activation. Results: Melatonin increases the expression of Fas and its ligand Fas L, this increase being responsible for cell death induced by the indolamine. Melatonin also produces a transient increase in intracellular oxidants and activation of the redox-regulated transcription factor Nuclear factor-kappaB. Inhibition of such activation prevents cell death and Fas/Fas L upregulation. Cytotoxic effect and Fas/Fas L regulation occur in all Ewing's cell lines studied, and do not occur in the other tumour cell lines studied where melatonin does not induce cell death. Conclusion: Our data offers new insights in the study of alternative therapeutic strategies in the treatment of Ewing's sarcoma. Further attention deserves to be given to the differences in the cellular biology of sensitive tumours that could explain the cytotoxic effect of melatonin and the increase in the level of free radicals caused by this molecule, in particular cancer types.

Melatonin is an endogenous indolamine, classically known as a light/dark regulator. Besides classical functions, melatonin has also showed to have a wide range of antitumoral effects in numerous cancer experimental models. However, no definite mechanism has been described to explain the whole range of antineoplasic effects. Here we describe a dual effect of melatonin on intracellular redox state in relation to its antiproliferative vs cytotoxic actions in cancer cells. Thus, inhibition of proliferation correlates with a decrease on intracellular reactive oxygen species (ROS) and increase of antioxidant defences (antioxidant enzymes and intracellular gluthation,GSH levels), while induction of cell death correlates with an increase on intracellular ROS and decrease of antioxidant defences. Moreover, cell death can be prevented by other well-known antioxidants or can be increased by hydrogen peroxide. Thus, tumour cell fate will depend on the ability of melatonin to induce either an antioxidant environment--related to the antiproliferative effect or a prooxidant environment related to the cytotoxic effect.

Incorporation of new therapeutic agents remains as a major challenge for treatment of patients with malignant haematological disorders. Melatonin is an indolamine without relevant side effects. It has been shown previously to exhibit synergism with several chemotherapeutic drugs in Ewing sarcoma cells by potentiating the extrinsic pathway of apoptosis. It also sensitizes human glioma cells against TRAIL by increasing DR5 expression. Here, we report the induction of cell death by melatonin in several human malignant haematological cell lines through the activation of the extrinsic pathway of apoptosis. Such activation was mediated by the increase in the expression of the death receptors Fas, DR4 and DR5 and their ligands Fas L and TRAIL, with a remarkable rise in the expression of Fas and Fas L. The cytotoxic effect and the increase in Fas and Fas L were dependent on Akt activation. Results were corroborated in blasts from bone marrow and peripheral blood of acute myeloid leukaemia patients, where melatonin induced cell death and increased both Fas and Fas L expressions. We conclude that melatonin may be considered as a potential antileukaemic agent and its therapeutic use, either alone or in combination with current chemotherapeutic drugs, should be taken into consideration for further research.

The availability of a rapidly increasing number of genome sequences of basidiomycete fungi has resulted in a strongly growing interest in basidiomycete genes and enzymes, in particularly those related to plant biomass degradation. The arsenal of enzymes produced by basidiomycete fungi differs markedly from those produced by ascomycete fungi, and the properties of the enzymes are also different. Most basidiomycetes do not have efficient transformation systems and large scale enzyme production in basidiomycetes is challenging. Therefore, heterologous production in ascomycete hosts is often the approach of choice, but there are still many challenges in producing these enzymes. Factors hampering heterologous expression of basidiomycete genes in ascomycetes include differences in the gene content such as in codon usage bias, and intron structure and size. In addition, basidiomycete enzymes are often considered foreign by the ascomycete production systems and therefore targets for their proteolytic systems, reducing the production yields.

Stand-alone virtual environments (VEs) using haptic devices have proved useful for assembly/disassembly simulation of mechanical components. Nowadays, collaborative haptic virtual environments (CHVEs) are also emerging. A new peer-to-peer collaborative haptic assembly simulator (CHAS) has been developed whereby two users can simultaneously carry out assembly tasks using haptic devices. Two major challenges have been addressed: virtual scene synchronization (consistency) and the provision of a reliable and effective haptic feedback. A consistency-maintenance scheme has been designed to solve the challenge of achieving consistency. Results show that consistency is guaranteed. Furthermore, a force-smoothing algorithm has been developed which is shown to improve the quality of force feedback under adverse network conditions. A range of laboratory experiments and several real trials between Labein (Spain) and Queen's University Belfast (Northern Ireland) have verified that CHAS can provide an adequate haptic interaction when both users perform remote assemblies (assembly of one user's object with an object grasped by the other user). Moreover, when collisions between grasped objects occur (dependent collisions), the haptic feedback usually provides satisfactory haptic perception. Based on a qualitative study, it is shown that the haptic feedback obtained during remote assemblies with dependent collisions can continue to improve the sense of co-presence between users with regard to only visual feedback.

The mechanisms underlying microRNA (miRNA) disruption in human disease are poorly understood. In cancer cells, the transcriptional silencing of tumor suppressor genes by CpG island promoter hypermethylation has emerged as a common hallmark. We wondered if the same epigenetic disruption can “hit” miRNAs in transformed cells. To address this issue, we have used cancer cells genetically deficient for the DNA methyltransferase enzymes in combination with a miRNA expression profiling. We have observed that DNA hypomethylation induces a release of miRNA silencing in cancer cells. One of the main targets is miRNA-124a, which undergoes transcriptional inactivation by CpG island hypermethylation in human tumors from different cell types. Interestingly, we functionally link the epigenetic loss of miRNA-124a with the activation of cyclin D kinase 6, a bona fide oncogenic factor, and the phosphorylation of the retinoblastoma, a tumor suppressor gene. [Cancer Res 2007;67(4):1424–9]

Glioblastoma multiforme (GBM) is an incurable malignancy with inherent tendency to recur. In this study, we have comparatively analyzed the epigenetic profile of 32 paired tumor samples of relapsed GBM and their corresponding primary neoplasms with special attention to genes involved in the mitochondria-independent apoptotic pathway. The CpG island promoter hypermethylation status was assessed by methylation-specific polymerase chain reaction and selected samples were double checked by bisulfite genomic sequencing. Thirteen genes were analyzed for DNA methylation: the pro-apoptotic CASP8, CASP3, CASP9, DcR1, DR4, DR5 and TMS1; the cell adherence CDH1 and CDH13; the candidate tumor suppressor RASSF1A and BLU; the cell cycle regulator CHFR and the DNA repair MGMT. The CpG island promoter hypermethylation profile of relapsed GBM in comparison with their corresponding primary tumors was identical in 37.5% of the cases, whereas in 62.5% of patients, differences in the DNA methylation patterns of the 13 genes were observed. The most prominent distinction was the presence of previously undetected CASP8 hypermethylation in the GBM relapses (P = 0.031). This finding was also linked to the observation that an unmethylated CASP8 CpG island together with methylated BLU promoter in the primary GBM was associated with prolonged time to tumor progression (P = 0.0035). Our data strongly suggest that hypermethylation of the pro-apoptotic CASP8 is a differential feature of GBM relapses. These remarkable findings may foster the development of therapeutic approaches using DNA demethylating drugs and activators of the extrinsic apoptotic pathway to improve the dismal prognosis of GBM.

Incorporation of new therapeutic agents remains as a major challenge for treatment of patients with malignant haematological disorders. Melatonin is an indolamine without relevant side effects. It has been shown previously to exhibit synergism with several chemotherapeutic drugs in Ewing sarcoma cells by potentiating the extrinsic pathway of apoptosis. It also sensitizes human glioma cells against TRAIL by increasing DR5 expression. Here, we report the induction of cell death by melatonin in several human malignant haematological cell lines through the activation of the extrinsic pathway of apoptosis. Such activation was mediated by the increase in the expression of the death receptors Fas, DR4 and DR5 and their ligands Fas L and TRAIL, with a remarkable rise in the expression of Fas and Fas L. The cytotoxic effect and the increase in Fas and Fas L were dependent on Akt activation. Results were corroborated in blasts from bone marrow and peripheral blood of acute myeloid leukaemia patients, where melatonin induced cell death and increased both Fas and Fas L expressions. We conclude that melatonin may be considered as a potential antileukaemic agent and its therapeutic use, either alone or in combination with current chemotherapeutic drugs, should be taken into consideration for further research.

The goal of this study was to evaluate different learning conditions for motor skill transfer. The study was divided into two experiments with the same task: learning a 3D trajectory with accuracy. The first experiment was focused on evaluating the efficiency of three feedback schemes for the target trajectory: visual, haptic and visual-haptic feedback. The second experiment was focused on analyzing the influence of decreasing the feedback during the training process. The results suggest that the best learning condition for learning a 3D trajectory with accuracy is to provide visual-haptic feedback, which facilitates the understanding of the dimension and orientation of each trajectory segment and solves any visual discrepancies that may exist. Furthermore, although continuous feedback can create dependences in users and impede the transfer of motor skills, feedback based on user request can also be dangerous since users can create a wrong mental representation that keep them from replicating the trajectory accurately. Therefore, when the performance of a task depends on references created during the training process, it seems appropriate for the system to provide automatic feedback based on user performance.

The concept of ion channels as membrane therapeutic targets and diagnostic/prognostic biomarkers has attracted growing attention. We therefore investigated the expression pattern and clinical significance of the Kv3.4 potassium channel subunit during the development and progression of head and neck squamous cell carcinomas (HNSCCs). KCNC4 mRNA levels were determined by real-time RT-PCR in both HNSCC tissue specimens and derived cell lines. Kv3.4 protein expression was evaluated by immunohistochemistry in paraffin-embedded tissue specimens from 84 patients with laryngeal/pharyngeal squamous cell carcinomas and 67 patients with laryngeal dysplasias. Molecular alterations were correlated with clinicopathological parameters and patient outcome. Increased KCNC4 mRNA levels were found in 15 (54%) of 28 tumours, compared to the corresponding normal epithelia and varied mRNA levels were detected in 12 HNSCC-derived cell lines analysed. Increased Kv3.4 protein expression was observed in 34 (40%) of 84 carcinomas and also at early stages of HNSCC tumourigenesis. Thus, 35 (52%) of 67 laryngeal lesions displayed Kv3.4-positive staining in the dysplastic areas, whereas both stromal cells and normal adjacent epithelia exhibited negligible expression. No significant correlations were found between Kv3.4-positive expression in HNSCC and clinical data; however, Kv3.4 expression tended to diminish in advanced-stage tumours. Interestingly, patients carrying Kv3.4-positive dysplasias experienced a significantly higher laryngeal cancer incidence than did those with negative lesions (p = 0.0209). In addition, functional studies using HNSCC cells revealed that inhibition of Kv3.4 expression by siRNA leads to the inhibition of cell proliferation via selective cell cycle arrest at the G2/M phase without affecting apoptosis. Collectively, these data demonstrate for the first time that Kv3.4 expression is frequently increased during HNSCC tumourigenesis and correlated significantly with a higher cancer risk. Our findings support a role for Kv3.4 in malignant transformation and provide original evidence for the potential clinical utility of Kv3.4 expression as a biomarker for cancer risk assessment.

Ewing sarcoma, the second most frequent bone cancer type, affects mainly adolescents, who have a survival of 50% 5 yr after diagnosis. Current treatments include a combination of surgery, radiotherapy and chemotherapy, which present potential serious side effects. Melatonin, a natural molecule without relevant side effects, has been previously shown to induce cytotoxicity in SK-N-MC cells, a Ewing sarcoma cell line. Here, we found that there is a synergy in the antitumor effect when melatonin (50 mum-1 mm) is combined with vincristine at the concentration of 5-10 nm or with ifosfamide at the range of 100 mum-1 mm. This synergism is due to the potentiation of cell death, particularly to the potentiation of apoptosis, i.e., mainly the extrinsic apoptotic pathway. There is a significant increase in the activation of caspase-3, -8, -9 and Bid when melatonin is combined with vincristine or ifosfamide compared to the individual treatments. Finally, there is also a potentiation of the early free radical production, likely dependent on the extrinsic apoptosis pathway activation, when the drugs are combined with melatonin. Other proteins which are related to this pathway including mitogen-activated protein kinase or protein kinase B/Akt are not involved in apoptosis induced by these agents separately or when combined. The results shown here together with the facts that: (i) no relevant side effects have been reported for melatonin and (ii) melatonin has a cytoprotective effect on noncancer cells, opens the door for a new approach in the treatment of the Ewing sarcoma family of tumors.

An undifferentiated status and the epigenetic inactivation of tumor-suppressor genes are hallmarks of transformed cells. Promoter CpG island hypermethylation of differentiating genes, however, has rarely been reported. The Groucho homologue Transducin-like Enhancer of Split 1 (TLE1) is a multitasked transcriptional corepressor that acts through the acute myelogenous leukemia 1, Wnt, and Notch signaling pathways. We have found that TLE1 undergoes promoter CpG island hypermethylation–associated inactivation in hematologic malignancies, such as diffuse large B-cell lymphoma and AML. We also observed a mutual exclusivity of the epigenetic alteration of TLE1 and the cytogenetic alteration of AML1. TLE1 reintroduction in hypermethylated leukemia/lymphoma cells causes growth inhibition in colony assays and nude mice, whereas TLE1-short hairpin RNA depletion in unmethylated cells enhances tumor growth. We also show that these effects are mediated by TLE1 transcriptional repressor activity on its target genes, such as Cyclin D1, Colony-Stimulating Factor 1 receptor, and Hairy/Enhancer of Split 1. These data suggest that TLE1 epigenetic inactivation contributes to the development of hematologic malignancies by disrupting critical differentiation and growth-suppressing pathways. [Cancer Res 2008;68(11):4116–22]

Virtual Environments using haptic devices have proved useful for assembly/disassembly simulation of mechanical components. To date most haptic virtual environments are stand-alone. Collaborative Haptic Virtual Environments (CHVEs) are distributed across a number of users via a network, such as the Internet. These present new challenges to the designer, such as consistency of the virtual environments, user-user haptic interaction, and scalability. The system described in this paper considers the CHVEs to be distributed over a packet-switched network such as the Internet. It gives priority to the validation of interactions between objects grasped by users; guarantees consistency across different users' virtual environments. The paper explains the components used and the consistency-maintenance scheme that guarantees the consistency of the virtual scene in the remote nodes. Consistency and force feedback results are also discussed. Results presented show the system maintains a consistent and satisfactory response when network incurs delay or packet jitter.