Epicatechin Stimulates Mitochondrial Activity and Selectively Sensitizes Cancer Cells to Radiation

Radiotherapy is the treatment of choice for solid tumors including pancreatic cancer, but the effectiveness of treatment is limited by radiation resistance. Resistance to chemotherapy or radiotherapy is associated with reduced mitochondrial respiration and drugs that stimulate mitochondrial respiration may decrease radiation resistance. The objectives of this study were to evaluate the potential of (-)-epicatechin to stimulate mitochondrial respiration in cancer cells and to selectively sensitize cancer cells to radiation. We investigated the natural compound (-)-epicatechin for effects on mitochondrial respiration and radiation resistance of pancreatic and glioblastoma cancer cells using a Clark type oxygen electrode, clonogenic survival assays, and Western blot analyses. (-)-Epicatechin stimulated mitochondrial respiration and oxygen consumption in Panc-1 cells. Human normal fibroblasts were not affected. (-)-Epicatechin sensitized Panc-1, U87, and MIA PaCa-2 cells with an average radiation enhancement factor (REF) of 1.7, 1.5, and 1.2, respectively. (-)-Epicatechin did not sensitize normal fibroblast cells to ionizing radiation with a REF of 0.9, suggesting cancer cell selectivity. (-)-Epicatechin enhanced Chk2 phosphorylation and p21 induction when combined with radiation in cancer, but not normal, cells. Taken together, (-)-epicatechin radiosensitized cancer cells, but not normal cells, and may be a promising candidate for pancreatic cancer treatment when combined with radiation

Inhibition of N-Methyl-D-aspartate-induced Retinal Neuronal Death by Polyarginine Peptides Is Linked to the Attenuation of Stress-induced Hyperpolarization of the Inner Mitochondrial Membrane Potential

It is widely accepted that overactivation of NMDA receptors, resulting in calcium overload and consequent mitochondrial dysfunction in retinal ganglion neurons, plays a significant role in promoting neurodegenerative disorders such as glaucoma. Calcium has been shown to initiate a transient hyperpolarization of the mitochondrial membrane potential triggering a burst of reactive oxygen species leading to apoptosis. Strategies that enhance cell survival signaling pathways aimed at preventing this adverse hyperpolarization of the mitochondrial membrane potential may provide a novel therapeutic intervention in retinal disease. In the retina, brain-derived neurotrophic factor has been shown to be neuroprotective, and our group previously reported a PSD-95/PDZ-binding cyclic peptide (CN2097) that augments brain-derived neurotrophic factor-induced pro-survival signaling. Here, we examined the neuroprotective properties of CN2097 using an established retinal in vivo NMDA toxicity model. CN2097 completely attenuated NMDA-induced caspase 3-dependent and -independent cell death and PARP-1 activation pathways, blocked necrosis, and fully prevented the loss of long term ganglion cell viability. Although neuroprotection was partially dependent upon CN2097 binding to the PDZ domain of PSD-95, our results show that the polyarginine-rich transport moiety C-R(7), linked to the PDZ-PSD-95-binding cyclic peptide, was sufficient to mediate short and long term protection via a mitochondrial targeting mechanism. C-R(7) localized to mitochondria and was found to reduce mitochondrial respiration, mitochondrial membrane hyperpolarization, and the generation of reactive oxygen species, promoting survival of retinal neurons

Regulation of Respiration and Apoptosis by Cytochrome c Threonine 58 Phosphorylation

Cytochrome c (cytc) is a multifunctional protein, acting as an electron carrier in the electron transport chain (ETC), where it shuttles electrons from bc1 complex to cytochrome c oxidase (COX), and as a trigger of type II apoptosis when released from the mitochondria. We previously showed that cytc is regulated in a highly tissue-specific manner: Cytc isolated from heart, liver, and kidney is phosphorylated on Y97, Y48, and T28, respectively. Here, we have analyzed the effect of a new Cytc phosphorylation site, threonine 58, which we mapped in rat kidney Cytc by mass spectrometry. We generated and overexpressed wild-type, phosphomimetic T58E, and two controls, T58A and T58I cytc; the latter replacement is found in human and testis-specific Cytc. In vitro, COX activity, caspase-3 activity, and heme degradation in the presence of H2o2 were decreased with phosphomimetic Cytc compared to wild-type. Cytc-knockout cells expressing T58E or T58I Cytc showed a reduction in intact cell respiration, mitochondrial membrane potential (∆Ψm), ROS production, and apoptotic activity compared to wild-type. We propose that, under physiological conditions, Cytc is phosphorylated, which controls mitochondrial respiration and apoptosis. Under conditions of stress Cytc phosphorylations are lost leading to maximal respiration rates, ∆Ψm hyperpolarization, ROS production, and apoptosis

Tissue‐specific regulation of cytochrome c by post‐translational modifications: respiration, the mitochondrial membrane potential, ROS, and apoptosis

Cytochrome c (Cytc) plays a vital role in the mitochondrial electron transport chain (ETC). In addition, it is a key regulator of apoptosis. Cytc has multiple other functions including ROS production and scavenging, cardiolipin peroxidation, and mitochondrial protein import. Cytc is tightly regulated by allosteric mechanisms, tissue‐specific isoforms, and post‐translational modifications (PTMs). Distinct residues of Cytc are modified by PTMs, primarily phosphorylations, in a highly tissue‐specific manner. These modifications downregulate mitochondrial ETC flux and adjust the mitochondrial membrane potential (ΔΨm), to minimize reactive oxygen species (ROS) production under normal conditions. In pathologic and acute stress conditions, such as ischemia–reperfusion, phosphorylations are lost, leading to maximum ETC flux, ΔΨm hyperpolarization, excessive ROS generation, and the release of Cytc. It is also the dephosphorylated form of the protein that leads to maximum caspase activation. We discuss the complex regulation of Cytc and propose that it is a central regulatory step of the mammalian ETC that can be rate limiting in normal conditions. This regulation is important because it maintains optimal intermediate ΔΨm, limiting ROS generation. We examine the role of Cytc PTMs, including phosphorylation, acetylation, methylation, nitration, nitrosylation, and sulfoxidation and consider their potential biological significance by evaluating their stoichiometry.—Kalpage, H. A., Bazylianska, V., Recanati, M. A., Fite, A., Liu, J., Wan, J., Mantena, N., Malek, M. H., Podgorski, I., Heath, E. I., Vaishnav, A., Edwards, B. F., Grossman, L. I., Sanderson, T. H., Lee, I., Hüttemann, M. Tissue‐specific regulation of cytochrome c by post‐translational modifications: respiration, the mitochondrial membrane potential, ROS, and apoptosis. FASEB J. 33, 1540–1553 (2019). www.fasebj.orgPeer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154496/1/fsb2fj201801417r.pd

Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney: Implications for AMP Kinase

Mammalian cytochrome c (Cytc) plays a key role in cellular life and death decisions, functioning as an electron carrier in the electron transport chain and as a trigger of apoptosis when released from the mitochondria. However, its regulation is not well understood. We show that the major fraction of Cytc iso- lated from kidneys is phosphorylated on Thr28, leading to a par- tial inhibition of respiration in the reaction with cytochrome c oxidase. To further study the effect of Cytc phosphorylation in vitro, we generated T28E phosphomimetic Cytc, revealing supe- rior behavior regarding protein stability and its ability to degrade reactive oxygen species compared with wild-type un- phosphorylated Cytc. Introduction of T28E phosphomimetic Cytc into Cytc knock-out cells shows that intact cell respiration, mitochondrial membrane potential (����m), and ROS levels are reduced compared with wild type. As we show by high resolu- tion crystallography of wild-type and T28E Cytc in combination with molecular dynamics simulations, Thr28 is located at a cen- tral position near the heme crevice, the most flexible epitope of the protein apart from the N and C termini. Finally, in silico prediction and our experimental data suggest that AMP kinase, which phosphorylates Cytc on Thr28 in vitro and colocalizes with Cytc to the mitochondrial intermembrane space in the kid- ney, is the most likely candidate to phosphorylate Thr28 in vivo. We conclude that Cytc phosphorylation is mediated in a tissue- specific manner and leads to regulation of electron transport chain flux via “controlled respiration,” preventing ����m hyperpolarization, a known cause of ROS and trigger of apoptosis

Role of Operon aaoSo-mutT in Antioxidant Defense in Streptococcus oligofermentans

Previously, we have found that an insertional inactivation of aaoSo, a gene encoding L-amino acid oxidase (LAAO), causes marked repression of the growth of Streptococcus oligofermentans. Here, we found that aaoSo and mutT, a homolog of pyrophosphohydrolase gene of Escherichia coli, constituted an operon. Deletion of either gene did not impair the growth of S. oligofermentans, but double deletion of both aaoSo and mutT was lethal. Quantitative PCR showed that the transcript abundance of mutT was reduced for 13-fold in the aaoSo insertional mutant, indicating that gene polarity derived from the inactivation of aaoSo attenuated the expression of mutT. Enzymatic assays were conducted to determine the biochemical functions of LAAO and MutT of S. oligofermentans. The results indicated that LAAO functioned as an aminoacetone oxidase [47.75 nmol H2O2 (min·mg protein)–1]; and MutT showed the pyrophosphohydrolase activity, which removed mutagens such as 8-oxo-dGTP. Like paraquat, aaoSo mutations increased the expression of SOD, and addition of aminoacetone (final concentration, 5 mM) decreased the mutant’s growth by 11%, indicating that the aaoSo mutants are under ROS stress. HPLC did reveal elevated levels of cytoplasmic aminoacetone in both the deletion and insertional gene mutants of aaoSo. Electron spin resonance spectroscopy showed increased hydroxyl radicals in both types of aaoSo mutant. This demonstrated that inactivation of aaoSo caused the elevation of the prooxidant aminoacetone, resulting the cellular ROS stress. Our study indicates that the presence of both LAAO and MutT can prevent endogenous metabolites-generated ROS and mutagens. In this way, we were able to determine the role of the aaoSo-mutT operon in antioxidant defense in S. oligofermentans

The effect of starch-based biomaterials on leukocyte adhesion and activation in vitro

Leukocyte adhesion to biomaterials has long been recognised as a key element to determine their inflammatory potential. Results regarding leukocyte adhesion and activation are contradictory in some aspects of the material’s effect in determining these events. It is clear that together with the wettability or hydrophilicity/hydrophobicity, the roughness of a substrate has a major effect on leukocyte adhesion. Both the chemical and physical properties of a material influence the adsorbed proteins layer which in turn determines the adhesion of cells. In this work polymorphonuclear (PMN) cells and a mixed population of monocytes/macrophages and lymphocytes (mononuclear cells) were cultured separately with a range of starch-based materials and composites with hydroxyapatite (HA). A combination of both reflected light microscopy and scanning electron microscopy (SEM) was used in order to study the leukocyte morphology. The quantification of the enzyme lactate dehydrogenase (LDH) was used to determine the number of viable cells adhered to the polymers. Cell adhesion and activation was characterised by immunocytochemistry based on the expression of several adhesion molecules, crucial in the progress of an inflammatory response. This work supports previous in vitro studies with PMN and monocytes/macrophages, which demonstrated that there are several properties of the materials that can influence and determine their biological response. From our study, monocytes/macrophages and lymphocytes adhere in similar amounts to more hydrophobic (SPCL) and to moderately hydrophilic (SEVA-C) surfaces and do not preferentially adhere to rougher substrates (SCA). Contrarily, more hydrophilic surfaces (SCA) induced higher PMN adhesion and lower activation. In addition, the hydroxyapatite reinforcement induces changes in cell behaviour for some materials but not for others. The observed response to starch-based biodegradable polymers was not significantly different from the control materials. Thus, the results reported herein indicate the low potential of the starch-based biodegradable polymers to induce inflammation especially the HA reinforced composite materials

Nanomaterials for Neural Interfaces

This review focuses on the application of nanomaterials for neural interfacing. The junction between nanotechnology and neural tissues can be particularly worthy of scientific attention for several reasons: (i) Neural cells are electroactive, and the electronic properties of nanostructures can be tailored to match the charge transport requirements of electrical cellular interfacing. (ii) The unique mechanical and chemical properties of nanomaterials are critical for integration with neural tissue as long-term implants. (iii) Solutions to many critical problems in neural biology/medicine are limited by the availability of specialized materials. (iv) Neuronal stimulation is needed for a variety of common and severe health problems. This confluence of need, accumulated expertise, and potential impact on the well-being of people suggests the potential of nanomaterials to revolutionize the field of neural interfacing. In this review, we begin with foundational topics, such as the current status of neural electrode (NE) technology, the key challenges facing the practical utilization of NEs, and the potential advantages of nanostructures as components of chronic implants. After that the detailed account of toxicology and biocompatibility of nanomaterials in respect to neural tissues is given. Next, we cover a variety of specific applications of nanoengineered devices, including drug delivery, imaging, topographic patterning, electrode design, nanoscale transistors for high-resolution neural interfacing, and photoactivated interfaces. We also critically evaluate the specific properties of particular nanomaterials—including nanoparticles, nanowires, and carbon nanotubes—that can be taken advantage of in neuroprosthetic devices. The most promising future areas of research and practical device engineering are discussed as a conclusion to the review.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/64336/1/3970_ftp.pd

ClimSim: A large multi-scale dataset for hybrid physics-ML climate emulation

Modern climate projections lack adequate spatial and temporal resolution due to computational constraints. A consequence is inaccurate and imprecise predictions of critical processes such as storms. Hybrid methods that combine physics with machine learning (ML) have introduced a new generation of higher fidelity climate simulators that can sidestep Moore's Law by outsourcing compute-hungry, short, high-resolution simulations to ML emulators. However, this hybrid ML-physics simulation approach requires domain-specific treatment and has been inaccessible to ML experts because of lack of training data and relevant, easy-to-use workflows. We present ClimSim, the largest-ever dataset designed for hybrid ML-physics research. It comprises multi-scale climate simulations, developed by a consortium of climate scientists and ML researchers. It consists of 5.7 billion pairs of multivariate input and output vectors that isolate the influence of locally-nested, high-resolution, high-fidelity physics on a host climate simulator's macro-scale physical state.The dataset is global in coverage, spans multiple years at high sampling frequency, and is designed such that resulting emulators are compatible with downstream coupling into operational climate simulators. We implement a range of deterministic and stochastic regression baselines to highlight the ML challenges and their scoring. The data (https://huggingface.co/datasets/LEAP/ClimSim_high-res) and code (https://leap-stc.github.io/ClimSim) are released openly to support the development of hybrid ML-physics and high-fidelity climate simulations for the benefit of science and society

How sulphate-reducing microorganisms cope with stress: lessons from systems biology

Sulphate-reducing microorganisms (SRMs) are a phylogenetically diverse group of anaerobes encompassing distinct physiologies with a broad ecological distribution. As SRMs have important roles in the biogeochemical cycling of carbon, nitrogen, sulphur and various metals, an understanding of how these organisms respond to environmental stresses is of fundamental and practical importance. In this Review, we highlight recent applications of systems biology tools in studying the stress responses of SRMs, particularly Desulfovibrio spp., at the cell, population, community and ecosystem levels. The syntrophic lifestyle of SRMs is also discussed, with a focus on system-level analyses of adaptive mechanisms. Such information is important for understanding the microbiology of the global sulphur cycle and for developing biotechnological applications of SRMs for environmental remediation, energy production, biocorrosion control, wastewater treatment and mineral recovery