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We have calculated the current density induced by an external magnetic field in a set of figure-eight-shaped expanded porphyrinoids. The studied octaphyrins can be divided into three classes (N2, N4, and N6) based on the number of the inner hydrogen atoms of the pyrrole rings. Using the Runge-Kutta method, the current density is split into diatropic and paratropic contributions that are analyzed separately. The calculations show that one common ring current consists of two rather independent pathways. Each of them follows the outer side of the molecular frame of one half of the molecule and passes to the inner side of the frame on the other half. The ring-current pathways are similar to the ones for [12]infinitene. However, the current density of the octaphyrins is though more complex having many branching points and pathways. Vertical through-space current-density pathways pass in the middle of the molecules through a plane that is parallel to the figure-eight-shaped view of the molecules when the magnetic field is perpendicular to the plane. The isolectronic N2 and the N4 dication sustain a weak paratropic ring current inside the molecule, which is also observed in the 1H NMR magnetic shielding constant of the inner hydrogen atoms. The diatropic current-density contribution dominates in the studied molecules. For the N4 and N6 molecules, the global current-density pathways are only diatropic and N6 sustains the strongest global diatropic current-density flux of 13.2 nA/T. We have calculated the current density induced by an external magnetic field in a set of figure-eight-shaped expanded porphyrinoids. The studied octaphyrins can be divided into three classes (N2, N4, and N6) based on the number of the inner hydrogen atoms of the pyrrole rings. Using the Runge-Kutta method, the current density is split into diatropic and paratropic contributions that are analyzed separately. The calculations show that one common ring current consists of two rather independent pathways. Each of them follows the outer side of the molecular frame of one half of the molecule and passes to the inner side of the frame on the other half. The ring-current pathways are similar to the ones for [12]infinitene. However, the current density of the octaphyrins is more complex having many branching points and pathways. Vertical through-space current-density pathways pass in the middle of the molecules through a plane that is parallel to the figure-eight-shaped view of the molecules when the magnetic field is perpendicular to the plane. The isolectronic N2 and the N4 dication sustain a weak paratropic ring current inside the molecule, which is also observed in the H-1 NMR magnetic shielding constant of the inner hydrogen atoms. The diatropic current-density contribution dominates in the studied molecules. For the N4 and N6 molecules, the global current-density pathways are only diatropic and N6 sustains the strongest global diatropic current-density flux of 13.2 nA T-1.

All animals must maintain genome and proteome integrity, especially when experiencing endogenous or exogenous stress. To cope, organisms have evolved sophisticated and conserved response systems: unfolded protein responses (UPRs) ensure proteostasis while DNA damage responses (DDRs) maintains genome integrity. Emerging evidence suggests that UPRs and DDRs crosstalk, but this remains poorly understood. Here, we demonstrate that depletion of the DNA primasespri-1orpri-2, which synthesize RNA primers at replication forks and whose inactivation causes DNA damage, activates the UPR of the endoplasmic reticulum (UPR-ER) inCaenorhabditis elegans, with especially strong activation in the germline. We observed activation of both the inositol-requiring-enzyme 1 (ire-1) and the protein kinase RNA-like ER kinase (pek-1) branches of the UPR-ER. Interestingly, activation of the UPR-ER output gene heat shock protein 4 (hsp-4) was partially independent of its canonical activators,ire-1and X-box binding protein (xbp-1), and instead required the third branch of the UPR-ER, activating transcription factor 6 (atf-6), suggesting functional redundancy. We further found that primase depletion specifically induces the UPR-ER, but not the distinct cytosolic or mitochondrial UPRs, suggesting that primase inactivation causes compartment-specific rather than global stress. Functionally, loss ofire-1orpek-1sensitized animals to replication stress caused by hydroxyurea. Finally, transcriptome analysis ofpri-1embryos revealed several deregulated processes that could cause UPR-ER activation, including protein glycosylation, calcium signaling, and fatty acid desaturation. Together, our data show that the UPR-ER, but not other UPRs, responds to replication fork stress and that the UPR-ER is required to alleviate this stress.

The micronutrient vitamin B12 is an essential cofactor for two enzymes: methionine synthase, which plays a key role in the one-carbon cycle; and methylmalonyl-CoA mutase, an enzyme in a pathway that breaks down branched-chain amino acids and odd-chain fatty acids. A second, vitamin B12-independent pathway that degrades methylmalonyl-CoA and its upstream metabolite propionic acid was recently described inCaenorhabditis elegans, the propionate shunt pathway. Activation of five shunt pathway genes in response to low vitamin B12 availability or high propionic acid levels is accomplished by a transcriptional regulatory mechanism involving two nuclear hormone receptors, NHR-10 and NHR-68. Here, we report that theC. elegansMediator subunitmdt-15is also essential for the activation of the propionate shunt pathway genes, likely by acting as a transcriptional coregulator for NHR-10.C. elegans mdt-15mutants fed a low vitamin B12 diet have transcriptomes resembling those of wild-type worms fed a high vitamin B12 diet, with low expression of the shunt genes. Phenotypically, the embryonic lethality ofmdt-15mutants is specifically rescued by diets high in vitamin B12, but not by dietary polyunsaturated fatty acids, which rescue many other phenotypes of themdt-15mutants. Finally, NHR-10 binds to MDT-15 in yeast-two-hybrid assays, and the transcriptomes ofnhr-10mutants resemble those ofmdt-15mutants. Our data show that MDT-15 is a key coregulator for an NHR regulating propionic acid detoxification, adding to roles played by NHR:MDT-15 partnerships in metabolic regulation and pinpointing vitamin B12 availability as a requirement formdt-15dependent embryonic development.

Mildly thermal air or HNO3 oxidized activated carbons catalyse oxidative dehydrogenative couplings of benzo[b]fused heteroaryl 2,2’‐dimers, e.g., 2‐(benzofuran‐2‐yl)‐1H‐indole, to chiral 3,3’‐coupled cyclooctatetraenes or carbazole‐type migrative products under O2 atmosphere. DFT calculations show that the radical cation and the Scholl‐type arenium cation mechanisms lead to different products with 2‐(benzofuran‐2‐yl)‐1H‐indole, being in accord with experimental product distributions., Oxidative coupling of benzofused heteroaryl dimers proceeded via divergent mechanistic routes depending on the used substrate and carbocatalyst. DFT level calculations using molecular quinones to compare the thermodynamics between radical and arenium cationic pathways rationalized the experimental behaviour of 2‐(benzofuran‐2‐yl)‐1H‐indole: HNO3 oxidized active carbon (AC) catalyst yielded carbazole derivative while air oxidized AC yielded COTs.

Caenorhabditis elegans has emerged as a major model in biomedical and environmental toxicology. Numerous papers on toxicology and pharmacology in C. elegans have been published, and this species has now been adopted by investigators in academic toxicology, pharmacology, and drug discovery labs. C. elegans has also attracted the interest of governmental regulatory agencies charged with evaluating the safety of chemicals. However, a major, fundamental aspect of toxicological science remains underdeveloped in C. elegans: xenobiotic metabolism and transport processes that are critical to understanding toxicokinetics and toxicodynamics, and extrapolation to other species. The aim of this review was to initially briefly describe the history and trajectory of the use of C. elegans in toxicological and pharmacological studies. Subsequently, physical barriers to chemical uptake and the role of the worm microbiome in xenobiotic transformation were described. Then a review of what is and is not known regarding the classic Phase I, Phase II, and Phase III processes was performed. In addition, the following were discussed (1) regulation of xenobiotic metabolism; (2) review of published toxicokinetics for specific chemicals; and (3) genetic diversity of these processes in C. elegans. Finally, worm xenobiotic transport and metabolism was placed in an evolutionary context; key areas for future research highlighted; and implications for extrapolating C. elegans toxicity results to other species discussed.

The electronic structure and stability of M '@M-n systems is considered using DFT. The metal atom M ' is a rare earth, actinide, or a d transition metal, and M is a coinage (Group 11) metal. The sigma aromaticity along the M-n ring is emphasized. As a further indicator for it, the magnetically induced currents along the ring are studied, and found to be considerably larger than the aromatic pi currents in benzene. A name saturnenes is suggested for these atom-inside-ring systems. Attention is also called for the known analogous stick-inside-ring systems.

Survival analysis is a technique to identify prognostic biomarkers and genetic vulnerabilities in cancer studies. Large-scale consortium-based projects have profiled >11,000 adult and >4,000 paediatric tumor cases with clinical outcomes and multi-omics approaches. This provides a resource for investigating molecular-level cancer etiologies using clinical correlations. Although cancers often arise from multiple genetic vulnerabilities and have deregulated gene sets (GSs), existing survival analysis protocols can report only on individual genes. Additionally, there is no systematic method to connect clinical outcomes with experimental (cell line) data. To address these gaps, we developed cSurvival (https://tau.cmmt.ubc.ca/cSurvival). cSurvival provides a user-adjustable analytical pipeline with a curated, integrated database, and offers three main advances: (a) joint analysis with two genomic predictors to identify interacting biomarkers, including new algorithms to identify optimal cutoffs for two continuous predictors; (b) survival analysis not only at the gene, but also the GS level; and (c) integration of clinical and experimental cell line studies to generate synergistic biological insights. To demonstrate these advances, we report three case studies. We confirmed findings of autophagy-dependent survival in colorectal cancers and of synergistic negative effects between high expression of SLC7A11 and SLC2A1 on outcomes in several cancers. We further used cSurvival to identify high expression of the Nrf2-antioxidant response element pathway as a main indicator for lung cancer prognosis and for cellular resistance to oxidative stress-inducing drugs. Together, these analyses demonstrate cSurvival’s ability to support biomarker prognosis and interaction analysis via gene- and GS-level approaches and to integrate clinical and experimental biomedical studies.Key pointsWe developed cSurvival, an advanced framework using clinical correlations to study biomarker interactions in cancers, with source code and curated datasets freely available for allcSurvival includes new algorithms to identify optimal cutoffs for two continuous predictors to stratify patients into risk groups, enabling for the first time joint analysis with two genomic predictors;cSurvival allows survival analysis at the gene set (GS) level with comprehensive and up-to-date GS librariesThe cSurvival pipeline integrates clinical outcomes and experimental cancer cell line data to generate synergistic biological insights and to mine for appropriate preclinical cell line toolscSurvival is built on a manually curated cancer outcomes database

Transcriptome profiling is essential for gene regulation studies in development and disease. Current web-based tools enable functional characterization of transcriptome data, but most are restricted to applying gene-list-based methods to single datasets, inefficient in leveraging up-to-date and species-specific information, and limited in their visualization options. Additionally, there is no systematic way to explore data stored in the largest transcriptome repository, NCBI GEO. To fill these gaps, we have developed eVITTA (easy Visualization and Inference Toolbox for Transcriptome Analysis; https://tau.cmmt.ubc.ca/eVITTA/). eVITTA provides modules for analysis and exploration of studies published in NCBI GEO (easyGEO), detailed molecular- and systems-level functional profiling (easyGSEA), and customizable comparisons among experimental groups (easyVizR). We tested eVITTA on transcriptomes of SARS-CoV-2 infected human nasopharyngeal swab samples, and identified a downregulation of olfactory signal transducers, in line with the clinical presentation of anosmia in COVID-19 patients. We also analyzed transcriptomes of Caenorhabditis elegans worms with disrupted S-adenosylmethionine metabolism, confirming activation of innate immune responses and feedback induction of one-carbon cycle genes. Collectively, eVITTA streamlines complex computational workflows into an accessible interface, thus filling the gap of an end-to-end platform capable of capturing both broad and granular changes in human and model organism transcriptomes., Graphical Abstract Graphical AbstractThe eVITTA toolbox provides an integrated graphical workflow to analyze, interpret and compare transcriptome data either extracted from published studies on NCBI GEO, or generated from new profiling experiments.

The model organism Caenorhabditis elegans mounts transcriptional defense responses against intestinal bacterial infections that elicit overlapping starvation and infection responses, the regulation of which is not well understood. Direct comparison of C. elegans that were starved or infected with Staphylococcus aureus revealed a large infection-specific transcriptional signature, which was almost completely abrogated by deletion of transcription factor hlh-30/TFEB, except for six genes including a flavin-containing monooxygenase (FMO) gene, fmo-2/FMO5. Deletion of fmo-2/FMO5 severely compromised infection survival, thus identifying the first FMO with innate immunity functions in animals. Moreover, fmo-2/FMO5 induction required the nuclear hormone receptor, NHR-49/PPAR-α, which controlled host defense cell non-autonomously. These findings reveal an infection-specific host response to S. aureus, identify HLH-30/TFEB as its main regulator, reveal FMOs as important innate immunity effectors in animals, and identify the mechanism of FMO regulation through NHR-49/PPAR-α during S. aureus infection, with implications for host defense and inflammation in higher organisms.

The response to insufficient oxygen (hypoxia) is orchestrated by the conserved hypoxia-inducible factor (HIF). However, HIF-independent hypoxia response pathways exist that act in parallel with HIF to mediate the physiological hypoxia response. Here, we describe a hypoxia response pathway controlled by

The response to insufficient oxygen (hypoxia) is orchestrated by the conserved Hypoxia-Inducible Factor (HIF). However, HIF-independent hypoxia response pathways exist that act in parallel to HIF to mediate the physiological hypoxia response. Here, we describe a HIF-independent hypoxia response pathway controlled byCaenorhabditis elegansNuclear Hormone Receptor NHR-49, an orthologue of mammalian Peroxisome Proliferator-Activated Receptor alpha (PPARα). We show thatnhr-49is required for worm survival in hypoxia and is synthetic lethal withhif-1in this context, demonstrating that these factors act independently. RNA-seq analysis shows that in hypoxianhr-49regulates a set of genes that arehif-1-independent, including autophagy genes that promote hypoxia survival. We further show that Nuclear Hormone Receptornhr-67is a negative regulator and Homeodomain-interacting Protein Kinasehpk-1is a positive regulator of the NHR-49 pathway. Together, our experiments define a new, essential hypoxia response pathway that acts in parallel to the well-known HIF-mediated hypoxia response.

Caenorhabditis elegans has emerged as a major model in biomedical and environmental toxicology. Numerous papers on toxicology and pharmacology in C. elegans have been published, and this model has now been adopted by investigators in academic toxicology, pharmacology, and drug discovery labs. C. elegans has also attracted the interest of governmental regulatory agencies charged with evaluating the safety of chemicals. However, a major, fundamental aspect of toxicological science remains underdeveloped in C. elegans: xenobiotic metabolism and transport processes that are critical to understanding toxicokinetics and toxicodynamics, and extrapolating to other species. The aim of this review was to initially briefly describe the history and trajectory of the use of C. elegans in toxicological and pharmacological studies. Subsequently, physical barriers to chemical uptake and role of the worm microbiome in xenobiotic transformation were described. Then a review on what is and is not known regarding the classic Phase I, Phase II, and Phase III processes was examined. In addition, the following were discussed (1) regulation of xenobiotic metabolism; (2) review of published toxicokinetics for specific chemicals; and (3) genetic diversity of these processes in C. elegans. Finally, worm xenobiotic transport and metabolism was placed in an evolutionary context; key areas for future research highlighted; and implications for extrapolating C. elegans toxicity results to other species discussed.

Ho et al. identified pathways, beyond lipid metabolism, that are required to maintain ER integrity and, when disrupted, activate the UPR by lipid bilayer stress through a sensor in Ire1. The resulting downstream transcriptional program differs from proteotoxic stress-induced UPR., Membrane integrity at the endoplasmic reticulum (ER) is tightly regulated, and its disturbance is implicated in metabolic diseases. Using an engineered sensor that activates the unfolded protein response (UPR) exclusively when normal ER membrane lipid composition is compromised, we identified pathways beyond lipid metabolism that are necessary to maintain ER integrity in yeast and in C. elegans. To systematically validate yeast mutants that disrupt ER membrane homeostasis, we identified a lipid bilayer stress (LBS) sensor in the UPR transducer protein Ire1, located at the interface of the amphipathic and transmembrane helices. Furthermore, transcriptome and chromatin immunoprecipitation analyses pinpoint the UPR as a broad-spectrum compensatory response wherein LBS and proteotoxic stress deploy divergent transcriptional UPR programs. Together, these findings reveal the UPR program as the sum of two independent stress responses, an insight that could be exploited for future therapeutic intervention., Graphical Abstract

Zinc is essential for cellular functions as it is a catalytic and structural component of many proteins. In contrast, cadmium is not required in biological systems and is toxic. Zinc and cadmium levels are closely monitored and regulated as their excess causes cell stress. To maintain homeostasis, organisms induce metal detoxification gene programs through stress responsive transcriptional regulatory complexes. In Caenorhabditis elegans, the MDT-15 subunit of the evolutionarily conserved Mediator transcriptional coregulator is required to induce genes upon exposure to excess zinc and cadmium. However, the regulatory partners of MDT-15 in this response, its role in cellular and physiological stress adaptation, and the putative role for mammalian MED15 in the metal stress responses remain unknown. Here, we show that MDT-15 interacts physically and functionally with the Nuclear Hormone Receptor HIZR-1 to promote molecular, cellular, and organismal adaptation to cadmium and excess zinc. Using gain- and loss-of-function mutants and qRT-PCR and reporter analysis, we find that mdt-15 and hizr-1 cooperate to induce zinc and cadmium responsive genes. Moreover, the two proteins interact physically in yeast-two-hybrid assays and this interaction is enhanced by the addition of zinc or cadmium, the former a known ligand of HIZR-1. Functionally, mdt-15 and hizr-1 mutants show defective storage of excess zinc in the gut and are hypersensitive to zinc-induced reductions in egg-laying. Furthermore, mdt-15 but not hizr-1 mutants are hypersensitive to cadmium-induced reductions in egg-laying, suggesting potential divergence of regulatory pathways. Lastly, mammalian MDT-15 orthologs bind genomic regulatory regions of metallothionein and zinc transporter genes in a cadmium and zinc-stimulated fashion, and human MED15 is required to induce a metallothionein gene in lung adenocarcinoma cells exposed to cadmium. Collectively, our data show that mdt-15 and hizr-1 cooperate to regulate cadmium detoxification and zinc storage and that this mechanism is at least partially conserved in mammals., Author summary In all life forms, zinc plays essential cellular and molecular roles. However, as with all molecules, excess zinc is hazardous, as is the presence of the physically and chemically highly similar cadmium. Organisms have evolved mechanisms to protect themselves against cadmium and high levels of zinc. Using the roundworm Caenorhabditis elegans, we previously showed that the Mediator subunit MDT-15 is essential in many cytoprotective responses including that against excess zinc or cadmium. Here, we find that the nuclear receptor HIZR-1, shown previously to convey high zinc detoxification, works together with MDT-15 to protect worms against high zinc as well as cadmium by cooperatively activating a cytoprotective gene expression program. Notably, the two proteins bind physically in a fashion that is enhanced by exogenous zinc or cadmium; these metals thus appear to act like stereotypical hormones by promoting a nuclear receptor–coregulator interaction. Exploring the evolutionary conservation of this mechanism, we find that MDT-15’s orthologues bind and regulate zinc and cadmium responsive genes in mammalian cell lines. This provides the first evidence that mammalian MED15 functions in stress response pathways, establishing it as a cytoprotective regulator across eukaryotes.

During bacterial infection, the host is confronted with multiple overlapping signals that are integrated at the organismal level to produce defensive host responses. How multiple infection signals are sensed by the host and how they elicit the transcription of host defense genes is much less understood at the whole-animal level than at the cellular level. The model organism Caenorhabditis elegans is known to mount transcriptional defense responses against intestinal bacterial infections that elicit overlapping starvation and infection responses, but the regulation of such responses is not well understood. Direct comparison of C. elegans that were starved or infected with Staphylococcus aureus revealed a large infection-specific transcriptional signature. This signature was almost completely abrogated by the deletion of transcription factor hlh-30/TFEB, except for six genes including a flavin-containing monooxygenase (FMO) gene, fmo-2/FMO5. Deletion of fmo-2/FMO5 severely compromised infection survival, thus identifying the first FMO with innate immunity functions in animals. Further, fmo-2/FMO5 was specifically induced by Gram-positive pathogens and Gram-positive natural microbiota of C. elegans. Moreover, the mechanism of fmo-2/FMO5 induction required the nuclear receptor, NHR-49/PPARα, which induced fmo-2/FMO5 and host defense cell non-autonomously. These findings for the first time reveal an infection-specific host response to S. aureus, identify HLH-30/TFEB as its main regulator, reveal that FMOs are important innate immunity effectors in animals, and identify the mechanism of FMO regulation through NHR-49/PPARα in C. elegans, with important implications for innate host defense in higher organisms.

Despite many advances, the molecular links between energy metabolism and longevity are not well understood. Here, we have used the nematode model Caenorhabditis elegans to study the role of the yet-uncharacterized gene R148.3 in fat accumulation and lifespan. In wild-type worms, a R148.3p::GFP reporter showed enhanced expression throughout life in the pharynx, in neurons, and in muscles. Functionally, a protein fusing a predicted 22 amino acid N-terminal signal sequence (SS) of R148.3 to mCherry displayed robust accumulation in coelomyocytes, indicating that R148.3 is a secreted protein. Systematic depletion of R148.3 by RNA interference (RNAi) at L1 but not at young-adult stage enhanced triglyceride accumulation, which was associated with increased food uptake and lower expression of genes involved in lipid oxidation. However, RNAi of R148.3 at both L1 and young-adult stages robustly diminished mean and maximal lifespan of wild-type worms, and also abolished the long-lived phenotypes of eat-2 and daf-2/InsR mutants. Based on these data, we propose that R148.3 is an SS that modulates fat mass and longevity in an independent manner.

Besides their technological applications, endohedral fullerenes provide ideal conditions for investigating molecular dynamics in restricted geometries. A representative of this class of systems, Sc3C2@C80 displays complex intramolecular dynamics. The motion of the 45Sc trimer has a remarkable effect on its electron paramagnetic resonance (EPR) spectrum, which changes from a symmetric 22-peak pattern at high temperature to a single broad lineshape at low temperature. The scandium trimer consists of two equivalent and one inequivalent metal atom, due to the carbon dimer rocking through the Sc3 triangle. We demonstrate through first-principles molecular dynamics (MD), EPR parameter tensor averaging, and spectral modelling that, at high temperatures, three-dimensional movement of the enclosed Sc3C2 moiety takes place, which renders the metal centers equivalent and their magnetic parameters effectively isotropic. In contrast, at low temperatures the dynamics becomes restricted to two dimensions within the equatorial belt of the Ih symmetric C80 host fullerene. This restores the inequivalence of the scandium centers and causes their anisotropic hyperfine couplings to broaden the experimental spectrum.

A 41-year-old Asian woman with bilateral renal angiomyolipomas (AML) was incidentally identified to have a balanced translocation, 46,XX,t(11;12)(p15.4;q15). She had no other features or family history to suggest a diagnosis of tuberous sclerosis. Her healthy daughter had the same translocation and no renal AML at the age of 3 years. Whole-genome sequencing was performed on genomic maternal DNA isolated from blood. A targeted de novo assembly was then conducted with ABySS for chromosomes 11 and 12. Sanger sequencing was used to validate the translocation breakpoints. As a result, genomic characterization of chromosomes 11 and 12 revealed that the 11p breakpoint disrupted the NUP98 gene in intron 1, causing a separation of the promoter and transcription start site from the rest of the gene. The translocation breakpoint on chromosome 12q was located in a gene desert. NUP98 has not yet been associated with renal AML pathogenesis, but somatic NUP98 alterations are recurrently implicated in hematological malignancies, most often following a gene fusion event. We also found evidence for complex structural events involving chromosome 12, which appear to disrupt the TDG gene. We identified a TDGP1 partially processed pseudogene at 12p12.1, which adds complexity to the de novo assembly. In conclusion, this is the first report of a germline constitutional structural chromosome rearrangement disrupting NUP98 that occurred in a generally healthy woman with bilateral renal AML.

SUMMARYMembrane integrity at the endoplasmic reticulum (ER) is tightly regulated and is implicated in metabolic diseases when compromised. Using an engineered sensor that exclusively activates the unfolded protein response (UPR) during aberrant ER membrane lipid composition, we identified pathways beyond lipid metabolism that are necessary to maintain ER integrity in yeast and are conserved in C. elegans. To systematically validate yeast mutants disrupting ER membrane homeostasis, we identified a lipid bilayer stress (LBS) sensing switch in the UPR transducer protein Ire1, located at the interface of the amphipathic and transmembrane helices. Furthermore, transcriptome and chromatin immunoprecipitation (ChIP) analyses pinpoint the UPR as a broad-spectrum compensatory pathway in which LBS and proteotoxic stress-induced UPR deploy divergent transcriptional programs. Together, these findings reveal the UPR program as the sum of two independent stress events and could be exploited for future therapeutic intervention.

Zinc is essential for cellular functions as it is a catalytic and structural component of many proteins. In contrast, cadmium is not required in biological systems and is toxic. Zinc and cadmium levels are closely monitored and regulated as their excess causes cell stress. To maintain homeostasis, organisms induce metal detoxification gene programs through stress responsive transcriptional regulatory complexes. In Caenorhabditis elegans, the MDT-15 subunit of the evolutionarily conserved Mediator transcriptional coregulator is required to induce genes upon exposure to excess zinc and cadmium. However, the regulatory partners of MDT-15 in this response, its role in cellular and physiological stress adaptation, and the putative role mammalian for MED15 in the metal stress responses remain unknown. Here, we show that MDT-15 interacts physically and functionally with the Nuclear Hormone Receptor HIZR-1 to promote molecular, cellular, and organismal adaptation to excess metals. Using gain- and loss-of-function mutants and qPCR and reporter analysis, we find that mdt-15 and hizr-1 cooperate to induce zinc and cadmium responsive genes. Moreover, the two proteins interact physically in yeast-two-hybrid assays and this interaction is enhanced by the addition of zinc or cadmium, the former a known ligand of HIZR-1. Functionally, mdt-15 and hizr-1 mutants show defective storage of excess zinc in the gut, and at the organismal level, mdt-15 mutants are hypersensitive to zinc- and cadmium-induced reductions in egg-laying. Lastly, mammalian MDT-15 orthologs bind genomic regulatory regions of metallothionein and zinc transporter genes in a metal-stimulated fashion, and human MED15 is required to induce a metallothionein gene in lung adenocarcinoma cells exposed to cadmium. Collectively, our data show that mdt-15 and hizr-1 cooperate to regulate metal detoxification and zinc storage and that this mechanism appears to be at least partially conserved in mammals.

Soluble oligomers of amyloid-β (Aβ) impair synaptic plasticity, perturb neuronal energy homeostasis, and are implicated in Alzheimer's disease (AD) pathogenesis. Therefore, significant efforts in AD drug discovery research aim to prevent the formation of Aβ oligomers or block their neurotoxicity. The eukaryotic elongation factor-2 kinase (eEF2K) plays a critical role in synaptic plasticity, and couples neurotransmission to local dendritic mRNA translation. Recent evidence indicates that Aβ oligomers activate neuronal eEF2K, suggesting a potential link to Aβ induced synaptic dysfunction. However, a detailed understanding of the role of eEF2K in AD pathogenesis, and therapeutic potential of eEF2K inhibition in AD, remain to be determined. Here, we show that eEF2K activity is increased in postmortem AD patient cortex and hippocampus, and in the hippocampus of aged transgenic AD mice. Furthermore, eEF2K inhibition using pharmacological or genetic approaches prevented the toxic effects of Aβ42 oligomers on neuronal viability and dendrite formation in vitro. We also report that eEF2K inhibition promotes the nuclear factor erythroid 2-related factor (NRF2) antioxidant response in neuronal cells, which was crucial for the beneficial effects of eEF2K inhibition in neurons exposed to Aβ42 oligomers. Accordingly, NRF2 knockdown or overexpression of the NRF2 inhibitor, Kelch-Like ECH-Associated Protein-1 (Keap1), significantly attenuated the neuroprotection associated with eEF2K inhibition. Finally, genetic deletion of the eEF2K ortholog efk-1 reduced oxidative stress, and improved chemotaxis and serotonin sensitivity in C. elegans expressing human Aβ42 in neurons. Taken together, these findings highlight the potential utility of eEF2K inhibition to reduce Aβ-mediated oxidative stress in AD.

Cell signaling pathways that control proliferation and determine cell fates are tightly regulated to prevent developmental anomalies and cancer. Transcription factors and coregulators are important effectors of signaling pathway output, as they regulate downstream gene programs. In Caenorhabditis elegans, several subunits of the Mediator transcriptional coregulator complex promote or inhibit vulva development, but pertinent mechanisms are poorly defined. Here, we show that Mediator’s dissociable cyclin dependent kinase 8 (CDK8) module (CKM), consisting of cdk-8, cic-1/Cyclin C, mdt-12/dpy-22, and mdt-13/let-19, is required to inhibit ectopic vulval cell fates downstream of the epidermal growth factor receptor (EGFR)-Ras-extracellular signal-regulated kinase (ERK) pathway. cdk-8 inhibits ectopic vulva formation by acting downstream of mpk-1/ERK, cell autonomously in vulval cells, and in a kinase-dependent manner. We also provide evidence that the CKM acts as a corepressor for the Ets-family transcription factor LIN-1, as cdk-8 promotes transcriptional repression by LIN-1. In addition, we find that CKM mutation alters Mediator subunit requirements in vulva development: the mdt-23/sur-2 subunit, which is required for vulva development in wild-type worms, is dispensable for ectopic vulva formation in CKM mutants, which instead display hallmarks of unrestrained Mediator tail module activity. We propose a model whereby the CKM controls EGFR-Ras-ERK transcriptional output by corepressing LIN-1 and by fine tuning Mediator specificity, thus balancing transcriptional repression vs. activation in a critical developmental signaling pathway. Collectively, these data offer an explanation for CKM repression of EGFR signaling output and ectopic vulva formation and provide the first evidence of Mediator CKM-tail module subunit crosstalk in animals.

Low temperatures slow aging and extend lifespan in many organisms, including Caenorhabditis elegans. However, the metabolic and homeostatic aspects of low temperature-induced longevity remain poorly understood. Here, we show that changes in lipid composition regulated by MDT-15/Mediator 15, transcriptional co-regulator, is essential for low temperature-induced longevity and proteostasis in C. elegans. We find that inhibition of mdt-15 prevents animals from living long at low temperatures. We show that MDT-15 up-regulates fat-7, a fatty acid desaturase, at low temperatures, which increases the ratio of unsaturated to saturated fatty acids. We further demonstrate that maintaining this increased fatty acid ratio is essential for protein homeostasis and longevity at low temperatures. Thus, the homeostasis of lipid composition by MDT-15 appears to be a limiting factor for C. elegans proteostasis and longevity at low temperatures. Our findings highlight the crucial roles of fat regulation in maintaining normal organismal physiology under different environmental conditions.