Connexin Trafficking And The Control Of Gap Junction Assembly In The Mouse Preimplantation Embryo

Gap junction assembly in the preimplantation mouse embryo is a temporally regulated event, beginning a few hours after the third cleavage during the morphogenetic event known as compaction. The purpose of the present study was to determine which connexins contribute to gap junctions in the preimplantation mouse embryo and how their assembly into gap junctions at compaction is regulated. Using antibodies raised against different synthetic C-terminal peptides of connexin43 (Cx43), this protein was shown to assemble into gap junction-like plaques beginning with compaction. Prior to this time no Cx43 can be detected in the plasma membrane. Cell fractionation and reverse transcription-polymerase chain reaction (RT-PCR) were employed to show that Cx43 mRNA is in polyribosomes at the 4-cell stage, suggesting that synthesis of Cx43 begins at least one cell cycle in advance of when gap junctions first form. The fate of nascent Cx43 was followed throughout preimplantation development by means of immunofluorescence in conjunction with confocal laser scanning microscopy. In morulae and blastocysts Cx43 becomes differentially distributed in the apposed plasma membranes: a zonular distribution predominates between outside blastomeres and trophectoderm cells whereas plaque-like localizations predominate between inside blastomeres and cells of the inner cell mass. The cytoplasmic immunoreactivity in morulae was deemed to be nascent connexin en route to the plasma membrane since it could be abolished by treatment with cycloheximide, and redistributed by treatment with monensin or brefeldin-A, known inhibitors of protein trafficking. These latter two drugs were shown to cause specific alterations in the structure and distribution of a variety of organelles in morulae, especially those involved in protein trafficking and degradation.;No conclusive evidence for the contribution of connexin32 (Cx32) to gap junctions in morulae could be found. However, using immunogold electron microscopy, the contribution of Cx43 was confirmed. Cx43 was also found to contribute to gap junctions in cumulus-oocyte complexes (COCs), both between cumulus granulosa cells and these cells and the oocyte. Although 100% of gap junctions between cumulus cells were labeled with Cx43 specific antisera, over 30% of gap junctions in morulae were not. Thus other members of the connexin gene family may yet be found to contribute to gap junctions at this stage. Treatment of uncompacted 8-cell embryos with either monensin or brefeldin-A inhibited the appearance of gap junction-like structures containing Cx43 and the onset of gap junctional coupling in a reversible manner. These data demonstrate that the onset of gap junction assembly during compaction is regulated post-translationally and involves mobilization of connexin43 and possibly other members of the connexin gene family through trafficking organelles to plasma membranes

Tyre recycling utilising molten metal – risk assessment of a laboratory scale investigation

This paper describes the process undertaken prior to the commencement of a proposed tyre laboratory scale experiment carried out in University College Cork, Ireland to identify the hazards, assess the risks, change the design and implement control measures to manage the hazards of the experiment

Sufficiency of hypoxia-inducible 2-oxoglutarate dioxygenases to block chemical oxidative stress-induced differentiation of human embryonic stem cells

Hypoxia benefits undifferentiated pluripotent stem cell renewal, and 2-oxoglutarate (2OG) dioxygenases have been implicated in pluripotent stem cell induction and renewal. We show in human embryonic stem cells (hESC) that an ambient oxygen-induced oxidative stress response elicited by culture in a hypoxic atmosphere (0.5% O2) correlates with the expression of 2OG dioxygenases, which oxidise DNA (TET1, 2, 3) and histone H3 (KDM4C), the former reflected by elevation in genomic 5-hydroxymethylcytosine (5hmC). siRNA-mediated targeting of KDM4C and TET1–3 induces hESC differentiation. Under ambient atmospheric oxygen (21% O2), exposure to a low inhibitory concentration of sodium arsenite (NaAsO2, IC10), as a model of chemically-induced oxidative stress, suppresses antioxidant gene expression, reduces mitochondrial membrane potential and induces hESC differentiation. Co-administration of the antioxidant N-acetyl-L-cysteine promoted anti-oxidant, pluripotency and 2OG dioxygenase gene expression, elevated genomic hydroxymethylation and blocked induction of differentiation. Transient ectopic expression of KDM4C or TET1 in ambient atmospheric oxygen achieved the same. Our study substantiates a role for 2OG-dependent dioxygenases in hypoxia's promotion of undifferentiated hESC self-renewal

Dielectrophoresis: A Review of Applications for Stem Cell Research

Dielectrophoresis can discriminate distinct cellular identities in heterogeneous populations, and monitor cell state changes associated with activation and clonal expansion, apoptosis, and necrosis, without the need for biochemical labels. Demonstrated capabilities include the enrichment of haematopoetic stem cells from bone marrow and peripheral blood, and adult stem cells from adipose tissue. Recent research suggests that this technique can predict the ultimate fate of neural stem cells after differentiation before the appearance of specific cell-surface proteins. This review summarises the properties of cells that contribute to their dielectrophoretic behaviour, and their relevance to stem cell research and translational applications

A tool for implementing privacy in Nano

© 2020 IEEE. Personal use of this material is permitted. Permission from IEEE must be obtained for all other uses, in any current or future media, including reprinting/republishing this material for advertising or promotional purposes, creating new collective works, for resale or redistribution to servers or lists, or reuse of any copyrighted component of this work in other works.We present a work in progress strategy for implementing privacy in Nano at the consensus level, that can be of independent interest. Nano is a cryptocurrency that uses an Open Representative Voting (ORV) as a consensus mechanism, a variant of Delegated Proof of Stake. Each transaction on the network is voted on by representatives, and each vote has a weight equal to the percentage of their total delegated balance. Every account can delegate their stake to any other account (including itself) and change it anytime it wants. The goal of this paper is to achieve a way for the consensus algorithm to function without knowing the individual balances of each account. The tool is composed of three different schemes. The first is a weighted threshold secret sharing scheme based on the Chinese Remainder Theorem for polynomial rings [1] and it's used to generate, in a distributed way, a secret that will be a private key of an additive ElGamal cryptosystem over elliptic curves (EC-EG) [2], which is additive homomorphic. The second scheme is the polynomials commitment scheme presented in [3] and is used to make the previous scheme verifiable, i.e., without the need of a trusted dealer. Finally, the third scheme is used to decrypt a ciphertext of the EC-EG cryptosystem without reconstructing the private key and, because of that, can be used multiple times.IEEEinfo:eu-repo/semantics/submittedVersio

A Study of Financial Analysis Expectations and Practices in the Engineering Management Workplace

This paper describes an on-going study of Master of Engineering Management (MEM) students and the financial analysis related job expectations and environment they face. The objective of this effort is to provide enhanced understanding of these requirements so that instructional content in the related courses can be focused to meet these needs. To achieve this goal, the study segments findings based on a range of organizational and job level characteristics to identify critical differences in the financial work environment and the financial tools that are employed. Preliminary findings are discussed in this paper and contrasts between public and private sector practices are examined

Approaching multiplet splitting in X-ray photoelectron spectra by density functional theory methods: NO and O2 molecules as examples

The ability of density functional theory (DFT) based methods to predict the multiplet splitting arising from the core hole ionization of molecules such as NO and O2, exhibiting an open shell grounds state, is explored. In the NO molecule, N(1s) or O(1s) ionization leads to 3Π and 1Π multiplets whereas for O2, the presence of an O(1s) core hole leads to doublet and quartet multiplets with distinct BEs. Multiplet splittings obtained using different exchange-correlation functionals show an overall good agreement with experiment and minor variations within the functionals studied when spin contamination resulting from unrestricted DFT calculations is accounted for

Differential many-body effects for initial and core-ion states: impact on XPS spectra

In this paper, the contribution of many body effects to the X-ray photoelectron spectroscopy, XPS, of an NO molecule are studied using wavefunction theory where the specific consequences of different many-body terms are examined and contrasted. It is shown that there is a differential importance of the many-body effects for the different configurations involved in the XPS. These are the ground, initial state configuration and final, N(1s) and O(1s) core-hole ionic configurations. The consequences of the many-body effects are examined for the binding energies, BEs, to the two final state multiplets, triplet and singlet, for each of the core ions and for the relative intensities of the XPS transitions to these multiplets. The many body effects examined are those described as static effects that arise for individual terms that are important. The objective is to understand the chemical and physical origins that determine the importance of the correlation effects for the XPS, rather than to obtain very accurate predictions of the BEs. An important theoretical construct that is tested and justified is the equivalent core approximation where the core ionized atom is replaced by the next higher element in the periodic table. This construct allows us to establish a correlation for the relative importance of the many-body effects in terms of effective charges of the different atoms. This is a correlation that has not been considered before and that we expect may have general relevance. The potential of the effects that we have identified for the XPS of NO to be relevant for the XPS of more complex, condensed phase systems is considered