The integrated stress response (ISR) attenuates the rate of protein synthesis while inducing expression of stress proteins in cells. Various insults activate kinases that phosphorylate the GTPase eIF2 leading to inhibition of its exchange factor eIF2B. Vanishing White Matter (VWM) is a neurological disease caused by eIF2B mutations that, like phosphorylated eIF2, reduce its activity. We show that introduction of a human VWM mutation into mice leads to persistent ISR induction in the central nervous system. ISR activation precedes myelin loss and development of motor deficits. Remarkably, long-term treatment with a small molecule eIF2B activator, 2BAct, prevents all measures of pathology and normalizes the transcriptome and proteome of VWM mice. 2BAct stimulates the remaining activity of mutant eIF2B complex in vivo, abrogating the maladaptive stress response. Thus, 2BAct-like molecules may provide a promising therapeutic approach for VWM and provide relief from chronic ISR induction in a variety of disease contexts.

Arrays suitable for genotoxicity screening are reported that generate metabolites from cytochrome P450 enzymes (CYPs) in thin-film spots. Array spots containing DNA, various human cyt P450s, and electrochemiluminescence (ECL) generating metallopolymer [Ru(bpy) 2 PVP 10 ] 2+ were exposed to H 2 O 2 to activate the enzymes. ECL from all spots was visualized simultaneously using a CCD camera. Using benzo [a]pyrene as a test substrate, enzyme activity for producing DNA damage in the arrays was found in the order CYP1B1 > CYP1A2 > CYP1A1 > CYP2E1 > myoglobin, the same as the order of their metabolic activity. Thus, these arrays estimate the relative propensity of different enzymes to produce genotoxic metabolites. This is the first demonstration of ECL arrays for highthroughput in vitro genotoxicity screening.Bioactivation of xenobiotic molecules by cytochrome P450 (cyt P450, or CYP) enzymes in the human liver is a major source of genotoxicity. Metabolites formed in this way can cause damage to genetic material. 1,2 Since levels of cyt P450 enzyme expression vary dramatically in different individuals, subpopulations may be subject to varying degrees of chemical or drug toxicity. 3 Thus, knowledge of which isoforms of cyt P450 produce toxic metabolites is critical in the development of new drugs, agricultural chemicals, and other substances that impact the public.Genotoxic metabolites and their nucleobase adducts can be detected by separation methods such as LC-MS. 4,5 These methods are very sensitive and provide specific and detailed molecular information, but may be limited for screening by throughput, analysis time, and cost. On the other hand, alternative, relatively rapid array technologies have been very successful in genomics and proteomics and are in principle capable of many thousands of measurements on a single chip. 6We recently demonstrated rapid, inexpensive, voltammetric genotoxicity screening sensors assembled from films of DNA and cyt P450s in single-electrode 7 and eight-electrode formats. 8 In a two-step process, test molecules are first bioactivated by the enzymes. Then, possible adducts with DNA nucleobases are detected by voltammetry using ruthenium tris(2,2′-bipyridyl)Ru II (Ru(bpy) 3 2+ ) to catalytically oxidize the guanine bases 9 in DNA. Nucleobase adducts formed as a consequence of the enzyme reaction in the enzyme/DNA films are not detected directly, but increases in voltammetric peaks result because the adducts cause DNA * To whom correspondence should be addressed. E-mail: james.rusling@uconn.edu. † University of Connecticut. ‡ University of Connecticut Health Center. [Ru(bpy) 2 (PVP) 10 ] 2+ contains six N-bonds to Ru, and is thought to produce ECL upon reaction with guanines in DNA according to the following pathway: NIH Public AccessECL generated from adsorbed polymer films is more efficient and intense compared to ECL produced by solution species. 15 The Ru II center is oxidized by the electrode (eq 1) and subsequently oxidizes a guanine in DNA to form a guanine radical (eq 2)...

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A fuel cell featuring two enzymes as electrocatalysts on specially modified electrodes-an O 2 -tolerant hydrogenase for the anode and bilirubin oxidase for the cathode-produces more than 0.1 mW cm À2 (electrode) at 1 V from a non-explosive hydrogen-air (80/20) mixture. The anode and cathode are prepared by covalently attaching the respective enzymes, via 1-pyrenebutyric acid functionalities, to multiwalled carbon nanotubes pre-coated on graphite. The fuel cell performance, which is limited by the reduction of O 2 (4%), represents an order-of-magnitude improvement over analogous cells in which the enzymes are conventionally adsorbed on graphite. These results bring forward realization of what is possible for niche devices able to generate small power outputs from gas mixtures under ambient conditions.

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The function of intestinal keratins is unknown, although keratin 8 (K8)–null mice develop colitis, hyperplasia, diarrhea, and mistarget jejunal apical markers. We quantified the diarrhea in K8-null stool and examined its physiologic basis. Isolated crypt-units from K8-null and wild-type mice have similar viability. K8-null distal colon has normal tight junction permeability and paracellular transport but shows decreased short circuit current and net Na absorption associated with net Cl secretion, blunted intracellular Cl/HCO3-dependent pH regulation, hyperproliferation and enlarged goblet cells, partial loss of the membrane-proximal markers H,K-ATPase-β and F-actin, increased and redistributed basolateral anion exchanger AE1/2 protein, and redistributed Na-transporter ENaC-γ. Diarrhea and protein mistargeting are observed 1–2 d after birth while hyperproliferation/inflammation occurs later. The AE1/2 changes and altered intracellular pH regulation likely account, at least in part, for the ion transport defects and hyperproliferation. Therefore, colonic keratins have a novel function in regulating electrolyte transport, likely by targeting ion transporters to their cellular compartments.

Cytochrome (cyt) P450s comprise the enzyme superfamily responsible for human oxidative metabolism of a majority of drugs and xenobiotics. Electronic delivery of electrons to cyt P450s could be used to drive the natural catalytic cycle for fundamental investigations, stereo-and regioselective synthesis, and biosensors. We describe herein nm-thick films on electrodes featuring excess human cyt P450s and cyt P450 reductase (CPR) microsomes that efficiently mimic the natural catalytic pathway for the first time. Redox potentials, electron-transfer rates, CO-binding, and substrate conversion rates confirmed that electrons are delivered from the electrode to CPR, which transfers them to cyt P450. The film system enabled electrochemical probing of the interaction between cyt P450 and CPR for the first time. Agreement of film voltammetry data with theoretical simulations support a pathway featuring a key equilibrium redox reaction in the natural catalytic pathway between reduced CPR and cyt P450 occurring within a CPR-cyt P450 complex uniquely poised for substrate conversion.

Platforms based on thin enzyme/DNA films were used in two-tier screening of chemicals for reactive metabolites capable of producing toxicity. Microsomes were used for the first time as sources of cytochrome (cyt) P450 enzymes in these devices. Initial rapid screening involved electrochemiluminescent (ECL) arrays featuring spots containing ruthenium poly(vinylpyridine), DNA, and rat liver microsomes or bicistronically expressed human cyt P450 2E1 (h2E1). Cyt P450 enzymes were activated via the NADPH/reductase cycle. When bioactivation of substrates in the films gives reactive metabolites, they are trapped by covalent attachment to DNA bases. The rate of increase in ECL with enzyme reaction time reflects relative DNA damage rates. “Toxic hits” uncovered by the array were studied in structural detail by using enzyme/DNA films on silica nanospheres as “nanoreactors” to provide nucleobase adducts from reactive metabolites. The utility of this synergistic approach was demonstrated by estimating relative DNA damage rates of three mutagenic N-nitroso compounds and styrene. Relative enzyme turnover rates for these compounds using ECL arrays and LC-UV-MS correlated well with TD50 values for liver tumor formation in rats. Combining ECL array and nanoreactor/LC−MS technologies has the potential for rapid, high-throughput, cost-effective screening for reactive metabolites and provides chemical structure information that is complementary to conventional toxicity bioassays.