Promotion of oxygen reduction by a bio-inspired tethered iron phthalocyanine carbon nanotube-based catalyst

Electrocatalysts for oxygen reduction are a critical component that may dramatically enhance the performance of fuel cells and metal-air batteries, which may provide the power for future electric vehicles. Here we report a novel bio-inspired composite electrocatalyst, iron phthalocyanine with an axial ligand anchored on single-walled carbon nanotubes, demonstrating higher electrocatalytic activity for oxygen reduction than the state-of-the-art Pt/C catalyst as well as exceptional durability during cycling in alkaline media. Theoretical calculations suggest that the rehybridization of Fe 3d orbitals with the ligand orbitals coordinated from the axial direction results in a significant change in electronic and geometric structure, which greatly increases the rate of oxygen reduction reaction. Our results demonstrate a new strategy to rationally design inexpensive and durable electrochemical oxygen reduction catalysts for metal-air batteries and fuel cells.close34

Establishment of Cell Lines Stably Expressing dCas9-Fusions to Address Kinetics of Epigenetic Editing

Epigenetic editing is a promising approach to modulate the local chromatin environment of target genes with the ultimate goal of stable gene expression reprogramming. Epigenetic editing tools minimally consist of a DNA-binding domain and an effector domain. The CRISPR/dCas9 platform, where mutations in the nuclease domains render the Cas9 protein inactive, is widely used to guide epigenetic effectors to their intended genomic loci. Its flexible nature, simple use, and relatively low cost have revolutionized the research field of epigenetic editing. Although effective expression modulation is readily achieved, only a few studies have addressed the maintenance of the induced effects on endogenous loci. Here, we describe a detailed protocol to engineer cells that stably express the CRISPR/dCas9-effectors. The protocol involves modification of published dCas9-based plasmid vectors for easy transfer of the effector domain between the vector designed for transient transfection and the vector used for establishing cell lines stably expressing the dCas9-effector fusion protein. Transient transfection of the dCas9-effector-producing cells with sgRNA-expressing plasmids allows studying of the maintenance of epigenetic editing. Targeting various genes in different chromatin contexts and/or co-targeting multiple CRISPR/dCas9-effectors can be used to unravel rules underlying maintained gene expression reprogramming

Attention to pain and fear of pain in patients with chronic pain

To investigate how acceptance of illness affects chronic pain in terms of attention towards pain and fearful thinking of pain. 62 participants (50 women) with chronic pain carried a palmtop computer for 2 weeks. Eight times each day auditory signals were delivered to cue participants to complete questions about their experience. Multilevel analyses indicated that on moments with more intense pain, more fearful thinking about pain, and less positive emotions, attention to pain was increased. Illness acceptance did not moderate the relation between pain intensity and attention to pain. Results further indicated that on moments with more intense pain, more negative emotions, and less positive emotions, fearful thinking about pain was increased. Of particular interest was the finding that the relationship between pain intensity and fearful thinking about pain was less strong for those high in acceptance. Pain captures attention and elicits fearful thinking about pain. Acceptance may be a useful avenue to lower negative thinking about pain, and to increase well-being in patients with chronic illnesses

Epigenetic editing:towards realization of the curable genome concept

Recent developments in biotechnology have enabled scientists to modulate DNA sequences in a precise way. These genome engineering technologies also open new possibilities to alter the epigenetic composition of the genome at any given genomic location thereby changing gene expression patterns, while leaving the primary DNA sequence intact. This new approach, so-called epigenetic editing, holds great promise to permanently reprogram cell identity. As reprogramming the epigenetic composition, and hence gene expression patterns is now technically feasible, the society needs to consider to what extent interference at the epigenetic level can be accepted. In this review, we discuss the potential epigenetic editing holds for research and therapy, and also touch upon societal implications of this rapidly growing research field

Functional Validation of the Putative Oncogenic Activity of PLAU

Plasminogen activator, urokinase (PLAU) is involved in cell migration, proliferation and tissue remodeling. PLAU upregulation is associated with an increase in aggressiveness, metastasis, and invasion of several cancer types, including breast cancer. In patients, this translates into decreased sensitivity to hormonal treatment, and poor prognosis. These clinical findings have led to the examination of PLAU as a biomarker for predicting breast cancer prognosis and therapy responses. In this study, we investigated the functional ability of PLAU to act as an oncogene in breast cancers by modulating its expression using CRISPR-deactivated Cas9 (CRISPR-dCas9) tools. Different effector domains (e.g., transcription modulators (VP64, KRAB)) alone or in combination with epigenetic writers (DNMT3A/3L, MSssI) were fused to dCas9 and targeted to the PLAU promoter. In MDA-MB-231 cells characterized by high PLAU expression downregulation of PLAU expression by CRISPR-dCas9-DNMT3A/3L-KRAB, resulted in decreased cell proliferation. Conversely, CRISPR-dCas9-VP64 induced PLAU upregulation in low PLAU expressing MCF-7 cells and significantly increased aggressiveness and invasion. In conclusion, modulation of PLAU expression affected metastatic related properties of breast cancer cells, thus further validating its oncogenic activity in breast cancer cells

KRAB-Induced Heterochromatin Effectively Silences PLOD2 Gene Expression in Somatic Cells and is Resilient to TGFβ1 Activation

Epigenetic editing, an emerging technique used for the modulation of gene expression in mammalian cells, is a promising strategy to correct disease-related gene expression. Although epigenetic reprogramming results in sustained transcriptional modulation in several in vivo models, further studies are needed to develop this approach into a straightforward technology for effective and specific interventions. Important goals of current research efforts are understanding the context-dependency of successful epigenetic editing and finding the most effective epigenetic effector(s) for specific tasks. Here we tested whether the fibrosis- and cancer-associated PLOD2 gene can be repressed by the DNA methyltransferase M.SssI, or by the non-catalytic Krüppel associated box (KRAB) repressor directed to the PLOD2 promoter via zinc finger- or CRISPR-dCas9-mediated targeting. M.SssI fusions induced de novo DNA methylation, changed histone modifications in a context-dependent manner, and led to 50%-70% reduction in PLOD2 expression in fibrotic fibroblasts and in MDA-MB-231 cancer cells. Targeting KRAB to PLOD2 resulted in the deposition of repressive histone modifications without DNA methylation and in almost complete PLOD2 silencing. Interestingly, both long-term TGFβ1-induced, as well as unstimulated PLOD2 expression, was completely repressed by KRAB, while M.SssI only prevented the TGFβ1-induced PLOD2 expression. Targeting transiently expressed dCas9-KRAB resulted in sustained PLOD2 repression in HEK293T and MCF-7 cells. Together, these findings point to KRAB outperforming DNA methylation as a small potent targeting epigenetic effector for silencing TGFβ1-induced and uninduced PLOD2 expression

Functional Validation of the Putative Oncogenic Activity of PLAU

Plasminogen activator, urokinase (PLAU) is involved in cell migration, proliferation and tissue remodeling. PLAU upregulation is associated with an increase in aggressiveness, metastasis, and invasion of several cancer types, including breast cancer. In patients, this translates into decreased sensitivity to hormonal treatment, and poor prognosis. These clinical findings have led to the examination of PLAU as a biomarker for predicting breast cancer prognosis and therapy responses. In this study, we investigated the functional ability of PLAU to act as an oncogene in breast cancers by modulating its expression using CRISPR-deactivated Cas9 (CRISPR-dCas9) tools. Different effector domains (e.g., transcription modulators (VP64, KRAB)) alone or in combination with epigenetic writers (DNMT3A/3L, MSssI) were fused to dCas9 and targeted to the PLAU promoter. In MDA-MB-231 cells characterized by high PLAU expression downregulation of PLAU expression by CRISPR-dCas9-DNMT3A/3L-KRAB, resulted in decreased cell proliferation. Conversely, CRISPR-dCas9-VP64 induced PLAU upregulation in low PLAU expressing MCF-7 cells and significantly increased aggressiveness and invasion. In conclusion, modulation of PLAU expression affected metastatic related properties of breast cancer cells, thus further validating its oncogenic activity in breast cancer cells.</p

Functional Validation of the Putative Oncogenic Activity of PLAU

Plasminogen activator, urokinase (PLAU) is involved in cell migration, proliferation and tissue remodeling. PLAU upregulation is associated with an increase in aggressiveness, metastasis, and invasion of several cancer types, including breast cancer. In patients, this translates into decreased sensitivity to hormonal treatment, and poor prognosis. These clinical findings have led to the examination of PLAU as a biomarker for predicting breast cancer prognosis and therapy responses. In this study, we investigated the functional ability of PLAU to act as an oncogene in breast cancers by modulating its expression using CRISPR-deactivated Cas9 (CRISPR-dCas9) tools. Different effector domains (e.g., transcription modulators (VP64, KRAB)) alone or in combination with epigenetic writers (DNMT3A/3L, MSssI) were fused to dCas9 and targeted to the PLAU promoter. In MDA-MB-231 cells characterized by high PLAU expression downregulation of PLAU expression by CRISPR-dCas9-DNMT3A/3L-KRAB, resulted in decreased cell proliferation. Conversely, CRISPR-dCas9-VP64 induced PLAU upregulation in low PLAU expressing MCF-7 cells and significantly increased aggressiveness and invasion. In conclusion, modulation of PLAU expression affected metastatic related properties of breast cancer cells, thus further validating its oncogenic activity in breast cancer cells.</p