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Two systems were studied using either a pump-probe spectroscopic technique or a density functional theory computational method. Ultrafast time-resolved infrared spectroscopy has been used to investigate the reactivity of V(CO)5, a doublet species, with silicon-hydrogen and carbon-hydrogen bonds. Using a ultraviolet pump pulse, a carbonyl was dissociated from V(CO)6 while dissolved in triethylsilane; subsequently, a broad bandwidth infrared pulse interrogated the system. On timescales less than 1 ns, vanadium pentacarbonyl does not appear to react. The second study used the Growing String Method incorporating density functional theory to examine the possible reaction mechanisms for the fluxional rearrangement of Fe3(CO)12 and Ru3(CO)12. Fluxional mechanisms have structurally identical reactants and products; the atoms swap positions with other atoms of the same element. The concerted bridge-opening, bridge-closing mechanism for the Fe3(CO)12 was calculated to have the lowest barrier of those tested at 0.777 kcal/mol; in this mechanism, all twelve carbonyl change position and the final structure is equivalent to the initial structure. At 4.285 kcal/mol, the next lowest energy barrier was calculated for the Cotton's Merry-Go-Round mechanism.

Supplemental material, PRISMA-2009-Checklist-MS-Word_1 for A systematic review of the effect of prior hypoglycaemia on cognitive function in type 1 diabetes by Suresh Rama Chandran, Peter Jacob and Pratik Choudhary in Therapeutic Advances in Endocrinology and Metabolism

Minichromosome maintenance protein 10 (Mcm10) is essential for eukaryotic DNA replication. Here, we describe compound heterozygous MCM10 mutations in patients with distinctive but overlapping clinical phenotypes – natural killer (NK) cell deficiency (NKD) and restrictive cardiomyopathy (RCM) with hypoplasia of the spleen and thymus. To understand the mechanism of Mcm10-associated disease, we modeled these mutations in human cell lines. Mcm10 deficiency causes chronic replication stress that reduces cell viability due to increased genomic instability and telomere erosion. Our data suggest that loss of Mcm10 function constrains telomerase activity by accumulating abnormal replication fork structures enriched with single-stranded DNA. Terminally-arrested replication forks in Mcm10-deficient cells require endonucleolytic processing by Mus81, as MCM10:MUS81 double mutants display decreased viability and accelerated telomere shortening. We propose that these bi-allelic mutations in MCM10 predispose specific cardiac and immune cell lineages to prematurely arrest during differentiation, causing the clinical phenotypes in both NKD and CM patients.

Complete and robust human genome duplication requires loading minichromosome maintenance (MCM) helicase complexes at many DNA replication origins, an essential process termed origin licensing. Licensing is restricted to G1 phase of the cell cycle, but G1 length varies widely among cell types. Using quantitative single-cell analyses, we found that pluripotent stem cells with naturally short G1 phases load MCM much faster than their isogenic differentiated counterparts with long G1 phases. During the earliest stages of differentiation toward all lineages, MCM loading slows concurrently with G1 lengthening, revealing developmental control of MCM loading. In contrast, ectopic Cyclin E overproduction uncouples short G1 from fast MCM loading. Rapid licensing in stem cells is caused by accumulation of the MCM loading protein, Cdt1. Prematurely slowing MCM loading in pluripotent cells not only lengthens G1 but also accelerates differentiation. Thus, rapid origin licensing is an intrinsic characteristic of stem cells that contributes to pluripotency maintenance.

SUMMARYThe authors find that human cells re-entering the cell cycle from quiescence have both an impaired p53-dependent DNA replication origin licensing checkpoint and slow origin licensing. This combination makes every first S phase underlicensed and hypersensitive to replication stress.ABSTRACTTo maintain tissue homeostasis, cells transition between cell cycle quiescence and proliferation. An essential G1 process is Minichromosome Maintenance complex (MCM) loading at DNA replication origins to prepare for S phase, known as origin licensing. A p53-dependent origin licensing checkpoint normally ensures sufficient MCM loading prior to S phase entry. We used quantitative flow cytometry and live cell imaging to compare MCM loading during the long first G1 upon cell cycle entry and the shorter G1 phases in the second and subsequent cycles. We discovered that despite the longer G1 phase, the first G1 after cell cycle re-entry is significantly underlicensed. As a result, the first S phase cells are hypersensitive to replication stress. This underlicensing is from a combination of slow MCM loading with a severely compromised origin licensing checkpoint. The hypersensitivity to replication stress increases over repeated rounds of quiescence. Thus, underlicensing after cell cycle re-entry from quiescence distinguishes a higher risk cell cycle that promotes genome instability.

KEAP1 is a substrate adaptor protein for a CUL3-based E3 ubiquitin ligase. Ubiquitylation and degradation of the antioxidant transcription factor NRF2 is considered the primary function of KEAP1; however, few other KEAP1 substrates have been identified. Because KEAP1 is altered in a number of human pathologies and has been proposed as a potential therapeutic target therein, we sought to better understand KEAP1 through systematic identification of its substrates. Toward this goal, we combined parallel affinity capture proteomics and candidate-based approaches. Substrate-trapping proteomics yielded NRF2 and the related transcription factor NRF1 as KEAP1 substrates. Our targeted investigation of KEAP1-interacting proteins revealed MCM3, an essential subunit of the replicative DNA helicase, as a new substrate. We show that MCM3 is ubiquitylated by the KEAP1-CUL3-RBX1 complex in cells and in vitro. Using ubiquitin remnant profiling, we identify the sites of KEAP1-dependent ubiquitylation in MCM3, and these sites are on predicted exposed surfaces of the MCM2–7 complex. Unexpectedly, we determined that KEAP1 does not regulate total MCM3 protein stability or subcellular localization. Our analysis of a KEAP1 targeting motif in MCM3 suggests that MCM3 is a point of direct contact between KEAP1 and the MCM hexamer. Moreover, KEAP1 associates with chromatin in a cell cycle-dependent fashion with kinetics similar to the MCM2–7 complex. KEAP1 is thus poised to affect MCM2–7 dynamics or function rather than MCM3 abundance. Together, these data establish new functions for KEAP1 within the nucleus and identify MCM3 as a novel substrate of the KEAP1-CUL3-RBX1 E3 ligase.

Cells initiate DNA replication at thousands of DNA replication origins every cell cycle. Minichromosome maintenance complexes (MCM) unwind DNA to initiate replication in S phase. MCM loading onto DNA, called “origin licensing”, occurs in G1 phase. Multiple mechanisms restrict origin licensing to G1 phase to prevent aberrant MCM loading and genotoxic re-replication in S phase. For this reason, cells load 5-10 fold excess MCM in G1 as dormant origins to protect against replication stress in S phase. The origin licensing checkpoint ensures sufficient MCM loading before S phase entry. MCM loading occurs in G1, yet cells have a variety of G1 lengths. Stem cells have short G1s while differentiated cells have long G1 phases. Additionally, cells may exit the cell cycle to quiescence, a state without division. Cells re-entering the cell cycle from quiescence have longer G1s than actively proliferating cells. How cells accomplish the same MCM loading under these different G1 lengths is poorly understood. We used quantitative single cell flow cytometry and live cell imaging to measure MCM loading across varying G1 lengths. We found that stem cells with short G1s load MCM significantly faster than differentiated cells with long G1s. MCM loading rate decreases and G1 length increases during stem cell differentiation to all germ layers. The rapid licensing is due to increased Cdt1 protein in stem cells. Rapid origin licensing is intrinsic to pluripotency, as slowing MCM loading in stem cells promotes their differentiation. Cells re-entering into long G1 phases from quiescence are underlicensed with less loaded MCM at S phase entry than proliferating cells. The underlicensing causes replication stress sensitivity in S phase, and repeated rounds of quiescence and re-entry increase the replication stress sensitivity. A defective origin licensing checkpoint combined with a slow MCM loading rate causes the underlicensing during cell cycle re-entry, and extending the first G1 phase rescues the underlicensing. Thus, cell cycle re-entry is a higher risk cell cycle than active proliferation. Proper regulation of the rate and amount of MCM loading is critical across different G1 types to control differentiation, proliferation and genome stability.

Complete and robust human genome duplication requires loading minichromosome maintenance (MCM) helicase complexes at many DNA replication origins, an essential process termed origin licensing. Licensing is restricted to G1 phase of the cell cycle, but G1 length varies widely among cell types. Using quantitative single-cell analyses, we found that pluripotent stem cells with naturally short G1 phases load MCM much faster than their isogenic differentiated counterparts with long G1 phases. During the earliest stages of differentiation toward all lineages, MCM loading slows concurrently with G1 lengthening, revealing developmental control of MCM loading. In contrast, ectopic Cyclin E overproduction uncouples short G1 from fast MCM loading. Rapid licensing in stem cells is caused by accumulation of the MCM loading protein, Cdt1. Prematurely slowing MCM loading in pluripotent cells not only lengthens G1 but also accelerates differentiation. Thus, rapid origin licensing is an intrinsic characteristic of stem cells that contributes to pluripotency maintenance., eLife digest From red blood cells to nerve cells, animals’ bodies contain many different types of specialized cells. These all begin as stem cells, which have the potential to divide and make more stem cells or to specialize. All dividing cells must first unwind their DNA so that it can be copied. To achieve this, cells load DNA-unwinding enzymes called helicases onto their DNA during the part of the cell cycle known as G1 phase. Cells must load enough helicase enzymes to ensure that their DNA is copied completely and in time. Stem cells divide faster than their specialized descendants, and have a much shorter G1 phase too. Yet these cells still manage to load enough helicases to copy their DNA. Little is known about how the amount, rate and timing of helicase loading varies between cells that divide at different speeds. Now Matson et al. have measured how quickly helicase enzymes are loaded onto DNA in individual human cells, including stem cells and specialized or “differentiated” cells. Stem cells loaded helicases rapidly to make up for the short time they spent in G1 phase, while differentiated cells loaded the enzymes more slowly. Measuring how the loading rate changed when stem cells were triggered to specialize showed that helicase loading slowed as the G1 phase got longer. Matson et al. found that the levels of key proteins required for helicase loading correlated with the rates of loading. Altering the levels of the proteins changed how quickly the enzymes were loaded and how the cells behaved – for example, slowing down the loading of helicases made the stem cells specialize quicker. These findings show that the processes of cell differentiation and DNA replication are closely linked. This study and future ones will help scientists understand what is happening during early animal development, when specialization first takes place, as well as what has gone wrong in cancer cells, which also divide quickly. A better understanding of this process also helps in regenerative medicine – where one of the challenges is to make enough specialized cells to transplant into a patient with tissue damage without those cells becoming cancerous.

This article draws on three theoretical fields, innovation intermediaries, socio-technical transitions and domestication studies to develop a process perspective of how user-side intermediary organisations seek to locally embed low carbon technologies. The term local embedding is increasingly used by transition researchers in a variety of ways. The first contribution of this paper is to explore and substantiate the concept of local embedding as the process of integrating technologies into local contexts of use. Intermediary organisations are conceived as contributing to local embedding where they facilitate, configure and broker change towards configurations that work. Nonetheless, understanding how these key intermediary processes relate as well as the influence of system dynamics on the work intermediaries undertake is still largely uncharted territory. The paper’s second contribution is a process perspective on the agency of intermediary organisations in local embedding. The resulting perspective offers insights into the agency of user-side intermediaries and later phases of transition processes.

All cells respond to osmotic stress by implementing molecular signaling events to protect the organism. Failure to properly adapt can lead to pathologies such as hypertension and ischemia-reperfusion injury. Mitogen-activated protein kinases (MAPKs) are activated in response to osmotic stress, as well as by signals acting through G protein-coupled receptors (GPCRs). For proper adaptation, the action of these kinases must be coordinated. To identify second messengers of stress adaptation, we conducted a mass spectrometry-based global metabolomics profiling analysis, quantifying nearly 300 metabolites in the yeast S. cerevisiae. We show that three branched-chain amino acid (BCAA) metabolites increase in response to osmotic stress and require the MAPK Hog1. Ectopic addition of these BCAA derivatives promotes phosphorylation of the G protein α subunit and dampens G protein-dependent transcription, similar to that seen in response to osmotic stress. Conversely, genetic ablation of Hog1 activity or the BCAA-regulatory enzymes leads to diminished phosphorylation of Gα and increased transcription. Taken together, our results define a new class of candidate second messengers that mediate cross talk between osmotic stress and GPCR signaling pathways., Author summary Just as organisms must adapt to a challenging environment, cells must respond to chemical or physical changes that occur within the organism. Regardless of the environmental cue, all cells use molecular signaling pathways to respond to those changes. Many are transmitted by G protein-coupled receptors (GPCRs) or the high osmolarity glycerol (HOG) pathway. While these pathways have been studied independently for decades, less is known about how they coordinate with each other to carry out the proper response, particularly when conflicting signals are present. One way coordination can be achieved is through “second messenger” molecules produced by one pathway to regulate another pathway. Here, we identify candidate second messengers of osmotic stress by global metabolite profiling analysis of the yeast S. cerevisiae. We find that three branched-chain amino acid (BCAA) metabolites increase in response to osmotic stress and require the stress response MAPK Hog1. We show that these BCAA derivatives are necessary and sufficient to recapitulate the effects of osmotic stress on the GPCR pathway. Our results identify a new way that HOG and GPCR pathways communicate, and may in the future guide better treatment strategies for stress-related cell damage.

To the best of our knowledge, two phosphorylation sites have been reported previously, among 11 known Vaccinia virus phosphoproteins. Here, via phosphopeptide mass spectrometry, up to 189 phosphorylation sites were identified among 48 proteins in preparations of purified Vaccinia mature virus (MV). 8.5% of phospho-residues were pTyr. Viral phosphoproteins were found in diverse functional classes, including structural proteins, membrane proteins and RNA polymerase subunits. Among the nine identified membrane phosphoproteins, the sites in just one, namely A14L, were deduced to be internal with respect to the accompanying membrane. Examination of sites in known substrates of the Vaccinia-encoded protein kinase VPK2, indicated VPK2 to be a proline-dependent kinase. The MV phosphoproteome was enriched in potential substrates of cellular kinases belonging to the CDK2/CDK3, CK2, and p38 groups. Quantitative mass spectrometry identified several sites that became phosphorylated during intravirion kinase activation in vitro, each showing one of two distinct pH-dependency profiles.