Observation of exsolution textures within Ba-Cu-O-rich solidified melts of Y-Ba-Cu-O materials and their relationship to Y123 nucleation and texturing

Layers (about 60-100 μm thick) of almost pure BaCuO2 (BC1), as determined using X-ray diffractometry (XRD) and scanning electron microscopy (SEM), coat the surfaces of YBa2Cu3O7-x (Y123) samples partial melt processed using a single-zone vertical furnace. The actual Cu/Ba ratio of the BC1 phase is 1.2-1.3 as determined using energy dispersive X-ray spectrometry (EDS). The nominally BC1 phase displays an exsolution of BC1.5 or BC2 in the form of thin plates (about 50-100 nm thick) along {100}-type cleavage planes or facets. The exsolved phase also fills cracks within the BC1 layer that require it to be in a molten state at some stage of processing. The samples were influenced by Pt contamination from the supporting wire, which may have stabilised the BC1.5 phase. Many of the Y123 grains have the same morphology as the exsolution domains, and run nearly parallel to the thin plates of the exsolved phases, strongly indicating that Y123 nucleation took place at the interface between the BC1 and the BC1.5 or BC2 exsolved phases. The network of nearly parallel exsolved 'channels' provides a matrix and a mechanism through which a high degree of local texture can be initiated in the material.

Auto-inhibition of hydrogen gas evolution on gold in aqueous acid solution

It was demonstrated recently that dramatic changes in the redox behaviour of gold/aqueous solution interfaces may be observed following either cathodic or thermal electrode pretreatment. Further work on the cathodic pretreatment of gold in acid solution revealed that as the activity of the gold surface was increased, its performance as a substrate for hydrogen gas evolution under constant potential conditions deteriorated. The change in activity of the gold atoms at the interface, which was attributed to a hydrogen embrittlement process (the occurrence of the latter was subsequently checked by surface microscopy), was confirmed, as in earlier work, by the appearance of a substantial anodic peak at ca. 0.5 V (RHE) in a post-activation positive sweep. Changes in the catalytic activity of a metal surface reflect the fact that the structure (or topography), thermodynamic activity and electronic properties of a surface are dependent not only on pretreatment but also, in the case of the hydrogen evolution reaction, vary with time during the course of reaction. As will be reported shortly, similar (and often more dramatic) time-dependent behaviour was observed for hydrogen gas evolution on other metal electrodes.

The active site behaviour of electrochemically synthesised gold nanomaterials

Even though gold is the noblest of metals, a weak chemisorber and is regarded as being quite inert, it demonstrates significant electrocatalytic activity in its nanostructured form. It is demonstrated here that nanostructured and even evaporated thin films of gold are covered with active sites which are responsible for such activity. The identification of these sites is demonstrated with conventional electrochemical techniques such as cyclic voltammetry as well as a large amplitude Fourier transformed alternating current (FT-ac) method under acidic and alkaline conditions. The latter technique is beneficial in determining if an electrode process is either Faradaic or capacitive in nature. The observed behaviour is analogous to that observed for activated gold electrodes whose surfaces have been severely disrupted by cathodic polarisation in the hydrogen evolution region. It is shown that significant electrochemical oxidation responses occur at discrete potential values well below that for the formation of the compact monolayer oxide of bulk gold and are attributed to the facile oxidation of surface active sites. Several electrocatalytic reactions are explored in which the onset potential is determined by the presence of such sites on the surface. Significantly, the facile oxidation of active sites is used to drive the electroless deposition of metals such as platinum, palladium and silver from their aqueous salts on the surface of gold nanostructures. The resultant surface decoration of gold with secondary metal nanoparticles not only indicates regions on the surface which are rich in active sites but also provides a method to form interesting bimetallic surfaces.

Large amplitude Fourier transformed AC voltammetric investigation of the active state electrochemistry of a copper/aqueous base interface and implications for electrocatalysis

The higher harmonic components available from large-amplitude Fourier-transformed alternating current (FT-ac) voltammetry enable the surface active state of a copper electrode in basic media to be probed in much more detail than possible with previously used dc methods. In particular, the absence of capacitance background current allows low-level Faradaic current contributions of fast electron-transfer processes to be detected; these are usually completely undetectable under conditions of dc cyclic voltammetry. Under high harmonic FT-ac voltammetric conditions, copper electrodes exhibit well-defined and reversible premonolayer oxidation responses at potentials within the double layer region in basic 1.0 M NaOH media. This process is attributed to oxidation of copper adatoms (Cu*) of low bulk metal lattice coordination numbers to surface-bonded, reactive hydrated oxide species. Of further interest is the observation that cathodic polarization in 1.0 M NaOH significantly enhances the current detected in each of the fundamental to sixth FT-ac harmonic components in the Cu*/Cu hydrous oxide electron-transfer process which enables the underlying electron transfer processes in the higher harmonics to be studied under conditions where the dc capacitance response is suppressed; the results support the incipient hydrous oxide adatom mediator (IHOAM) model of electrocatalysis. The underlying quasi-reversible interfacial Cu*/Cu hydrous oxide process present under these conditions is shown to mediate the reduction of nitrate at a copper electrode, while the mediator for the hydrazine oxidation reaction appears to involve a different mediator or active state redox couple. Use of FT-ac voltammetry offers prospects for new insights into the nature of active sites and electrocatalysis at the electrode/solution interface of Group 11 metals in aqueous media.

Reduction of Au3+ ions by activated surface atoms of platinum

In this work it is demonstrated that Pt electrodes can be activated by cathodic polarisation in the hydrogen evolution region which makes it prone to oxidation at potentials below that of bulk oxide formation. When an activated Pt electrode is placed in an aqueous HAuCl4 solution the electroless deposition of Au onto the surface of the electrode is observed and confirmed by cyclic voltammetry and XPS measurements. It is demonstrated that the oxidation of active Pt surface atoms provides the driving force for the spontaneous reduction of Au3+ ions into metallic Au to generate a Pt/Au surface which is highly active for the electro-oxidation of ethanol.

The electrochemical corrosion behaviour of quaternary gold alloys when exposed to 3.5% NaCl solution

Lower carat gold alloys, specifically 9 carat gold alloys, containing less than 40 % gold, and alloying additions of silver, copper and zinc, are commonly used in many jewellery applications, to offset high costs and poor mechanical properties associated with pure gold. While gold is considered to be chemically inert, the presence of active alloying additions raises concerns about certain forms of corrosion, particularly selective dissolution of these alloys. The purpose of this study was to systematically study the corrosion behaviour of a series of quaternary gold–silver–copper–zinc alloys using dc potentiodynamic scanning in saline (3.5 % NaCl) environment. Full anodic/cathodic scans were conducted to determine the overall corrosion characteristics of the alloy, followed by selective anodic scans and subsequent morphological and compositional analysis of the alloy surface and corroding media to determine the extent of selective dissolution. Varying degrees of selective dissolution and associated corrosion rates were observed after anodic polarisation in 3.5 % NaCl, depending on the alloy composition. The corrosion behaviour of the alloys was determined by the extent of anodic reactions which induce (1) formation of oxide scales on the alloy surface and or (2) dissolution of Zn and Cu species. In general, the improved corrosion characteristics of alloy #3 was attributed to the composition of Zn/Cu in the alloy and thus favourable microstructure promoting the formation of protective oxide/chloride scales and reducing the extent of Cu and Zn dissolution.

The molecular basis for selective inhibition of unconventional mRNA splicing by an IRE1-binding small molecule

IRE1 couples endoplasmic reticulum unfolded protein load to RNA cleavage events that culminate in the sequence-specific splicing of the Xbp1 mRNA and in the regulated degradation of diverse membrane-bound mRNAs. We report on the identification of a small molecule inhibitor that attains its selectivity by forming an unusually stable Schiff base with lysine 907 in the IRE1 endonuclease domain, explained by solvent inaccessibility of the imine bond in the enzyme-inhibitor complex. The inhibitor (abbreviated 4μ8C) blocks substrate access to the active site of IRE1 and selectively inactivates both Xbp1 splicing and IRE1-mediated mRNA degradation. Surprisingly, inhibition of IRE1 endonuclease activity does not sensitize cells to the consequences of acute endoplasmic reticulum stress, but rather interferes with the expansion of secretory capacity. Thus, the chemical reactivity and sterics of a unique residue in the endonuclease active site of IRE1 can be exploited by selective inhibitors to interfere with protein secretion in pathological settings.

The Arabidopsis thaliana double-stranded RNA binding protein DRB1 directs guide strand selection from microRNA duplexes

In Arabidopsis thaliana (Arabidopsis), DICER-LIKE1 (DCL1) functions together with the double-stranded RNA binding protein (dsRBP), DRB1, to process microRNAs (miRNAs) from their precursor transcripts prior to their transfer to the RNA-induced silencing complex (RISC). miRNA-loaded RISC directs RNA silencing of cognate mRNAs via ARGONAUTE1 (AGO1)-catalyzed cleavage. Short interefering RNAs (siRNAs) are processed from viral-derived or transgene-encoded molecules of doublestranded RNA (dsRNA) by the DCL/dsRBP partnership, DCL4/DRB4, and are also loaded to AGO1-catalyzed RISC for cleavage of complementary mRNAs. Here, we use an artificial miRNA (amiRNA) technology, transiently expressed in Nicotiana benthamiana, to produce a series of amiRNA duplexes with differing intermolecular thermostabilities at the 5′ end of duplex strands. Analyses of amiRNA duplex strand accumulation and target transcript expression revealed that strand selection (amiRNA and amiRNA*) is directed by asymmetric thermostability of the duplex termini. The duplex strand possessing a lower 59 thermostability was preferentially retained by RISC to guide mRNA cleavage of the corresponding target transgene. In addition, analysis of endogenous miRNA duplex strand accumulation in Arabidopsis drb1 and drb2345 mutant plants revealed that DRB1 dictates strand selection, presumably by directional loading of the miRNA duplex onto RISC for passenger strand degradation. Bioinformatic and Northern blot analyses of DCL4/DRB4-dependent small RNAs (miRNAs and siRNAs) revealed that small RNAs produced by this DCL/dsRBP combination do not conform to the same terminal thermostability rules as those governing DCL1/DRB1-processed miRNAs. This suggests that small RNA processing in the DCL1/DRB1-directed miRNA and DCL4/DRB4-directed sRNA biogenesis pathways operates via different mechanisms.

Hairpin RNAs derived from RNA polymerase II and polymerase III promoter-directed transgenes are processed differently in plants

RNA polymerase III (Pol III) as well as Pol II (35S) promoters are able to drive hairpin RNA (hpRNA) expression and induce target gene silencing in plants. siRNAs of 21 nt are the predominant species in a 35S Pol II line, whereas 24- and/or 22-nucleotide (nt) siRNAs are produced by a Pol III line. The 35S line accumulated the loop of the hpRNA, in contrast to full-length hpRNA in the Pol III line. These suggest that Pol II and Pol III-transcribed hpRNAs are processed by different pathways. One Pol III transgene produced only 24-nt siRNAs but silenced the target gene efficiently, indicating that the 24-nt siRNAs can direct mRNA degradation; specific cleavage was confirmed by 59 rapid amplification of cDNA ends (RACE). Both Pol II- and Pol III-directed hpRNA transgenes induced cytosine methylation in the target DNA. The extent of methylation is not correlated with the level of 21-nt siRNAs, suggesting that they are not effective inducers of DNA methylation. The promoter of a U6 transgene was significantly methylated, whereas the promoter of the endogenous U6 gene was almost free of cytosine methylation, suggesting that endogenous sequences are more resistant to de novo DNA methylation than are transgene constructs. Published by Cold Spring Harbor Laboratory Press. Copyright © 2008 RNA Society.

Efficient silencing of endogenous microRNAs using artificial microRNAs in Arabidopsis thaliana

We report here that the expression of endogenous microRNAs (miRNAs) can be efficiently silenced in Arabidopsis thaliana (Arabidopsis) using artificial miRNA (amiRNA) technology. We demonstrate that an amiRNA designed to target a mature miRNA directs silencing against all miRNA family members, whereas an amiRNA designed to target the stem-loop region of a miRNA precursor transcript directs silencing against only the individual family member targeted. Furthermore, our results indicate that amiRNAs targeting both the mature miRNA and stem-loop sequence direct RNA silencing through cleavage of the miRNA precursor transcript, which presumably occurs in the nucleus of a plant cell during the initial stages of miRNA biogenesis. This suggests that small RNA (sRNA)-guided RNA cleavage in plants occurs not only in the cytoplasm, but also in the nucleus. Many plant miRNA gene families have been identified via sequencing and bioinformatic analysis, but, to date, only a small tranche of these have been functionally characterized due to a lack of effective forward or reverse genetic tools. Our findings therefore provide a new and powerful reverse-genetic tool for the analysis of miRNA function in plants. © The Author 2010. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPP and IPPE, SIBS, CAS.

Plant and animal microRNAs : similarities and differences

Plant and animal microRNAs (miRNAs) are evolutionarily ancient small RNAs, ∼19-24 nucleotides in length, that are generated by cleavage from larger highly structured precursor molecules. In both plants and animals, miRNAs posttranscriptionally regulate gene expression through interactions with their target mRNAs, and these targets are often genes involved with regulating key developmental events. Despite these similarities, plant and animal miRNAs exert their control in fundamentally different ways. Generally, animal miRNAs repress gene expression by mediating translational attenuation through (multiple) miRNA-binding sites located within the 3′ untranslated region of the target gene. In contrast, almost all plant miRNAs regulate their targets by directing mRNA cleavage at single sites in the coding regions. These and other differences suggest that the two systems may have originated independently, possibly as a prerequisite to the development of complex body plans. © Springer-Verlag 2005.

The M(r) 43K major capsid protein of rice ragged stunt oryzavirus is a post-translationally processed product of a M(r) 67 348 polypeptide encoded by genome segment 8

The nucleotide sequence of DNA complementary to rice ragged stunt oryzavirus (RRSV) genome segment 8 (S8) of an isolate from Thailand was determined. RRSV S8 is 1 914 bp in size and contains a single large open reading frame (ORF) spanning nucleotides 23 to 1 810 which is capable of encoding a protein of M(r) 67 348. The N-terminal amino acid sequence of a ~43K virion polypeptide matched to that inferred for an internal region of the S8 coding sequence. These data suggest that the 43K protein is encoded by S8 and is derived by a proteolytic cleavage. Predicted polypeptide sizes from this possible cleavage of S8 protein are 26K and 42K. Polyclonal antibodies raised against a maltose binding protein (MBP)-S8 fusion polypeptide (expressed in Escherichia coli) recognised four RRSV particle associated polypeptides of M(r) 67K, 46K, 43K and 26K and all except the 26K polypeptide were also highly immunoreactive to polyclonal antibodies raised against purified RRSV particles. Cleavage of the MBP-S8 fusion polypeptide with protease Factor X produced the expected 40K MBP and two polypeptides of apparent M(r) 46K and 26K. Antibodies to purified RRSV particles reacted strongly with the intact fusion protein and the 46K cleavage product but weakly to the 26K product. Furthermore, in vitro transcription and translation of the S8 coding region revealed a post-translational self cleavage of the 67K polypeptide to 46K and 26K products. These data indicate that S8 encodes a structural polypeptide, the majority of which is auto- catalytically cleaved to 26K and 46K proteins. The data also suggest that the 26K protein is the self cleaving protease and that the 46K product is further processed or undergoes stable conformational changes to a ~43K major capsid protein.

Secretome and degradome profiling shows that Kallikrein-related peptidases 4, 5, 6, and 7 induce TGFβ-1 signaling in ovarian cancer cells

Kallikrein-related peptidases, in particular KLK4, 5, 6 and 7 (4-7), often have elevated expression levels in ovarian cancer. In OV-MZ-6 ovarian cancer cells, combined expression of KLK4-7 reduces cell adhesion and increases cell invasion and resistance to paclitaxel. The present work investigates how KLK4-7 shape the secreted proteome ("secretome") and proteolytic profile ("degradome") of ovarian cancer cells. The secretome comparison consistently identified >900 proteins in three replicate analyses. Expression of KLK4-7 predominantly affected the abundance of proteins involved in cell-cell communication. Among others, this includes increased levels of transforming growth factor β-1 (TGFβ-1). KLK4-7 co-transfected OV-MZ-6 cells share prominent features of elevated TGFβ-1 signaling, including increased abundance of neural cell adhesion molecule L1 (L1CAM). Augmented levels of TGFβ-1 and L1CAM upon expression of KLK4-7 were corroborated in vivo by an ovarian cancer xenograft model. The degradomic analysis showed that KLK4-7 expression mostly affected cleavage sites C-terminal to arginine, corresponding to the preference of kallikreins 4, 5 and 6. Putative kallikrein substrates include chemokines, such as growth differentiation factor 15 (GDF 15) and macrophage migration inhibitory factor (MIF). Proteolytic maturation of TGFβ-1 was also elevated. KLK4-7 have a pronounced, yet non-degrading impact on the secreted proteome, with a strong association between these proteases and TGFβ-1 signaling in tumor biology. © 2013 Federation of European Biochemical Societies.

Cellular stress triggers the human topoisomerase I damage response independently of DNA damage in a p53 controlled manner

The 'human topoisomerase I (htopoI) damage response' was reported to be triggered by various kinds of DNA lesions. Also, a high and persistent level of htopoI cleavage complexes correlated with apoptosis. In the present study, we demonstrate that DNA damage-independent induction of cell death using colcemid and tumor necrosis factor is also accompanied by a strong htopoI response that correlates with the onset of apoptotic hallmarks. Consequently, these results suggest that htopoI cleavage complex formation may be caused by signaling pathways independent of the kind of cellular stress. Thus, protein interactions or signaling cascades induced by DNA damage or cellular stress might lead to the formation of stabilized cleavage complexes rather than the DNA lesion itself. Finally, we show that p53 not only plays a key role in the regulation of the htopoI response to UV-C irradiation but also to treatment with colcemid.

The human topoisomerase I damage response plays a role in apoptosis

Previous studies have shown that human topoisomerase I cleavage complexes form as a response to various DNA damages in vivo, the so called human topoisomerase I “damage response”. It was suggested that this damage response may play a role in DNA repair as well as in apoptosis, but only very few investigations have been done and the significance of the damage response still remains unclear. Here we demonstrate that human topoisomerase I cleavage complexes induced by high doses of UV irradiation are highly stable for up to 48 h. Furthermore, we show that human topoisomerase I cleavage complexes correlate with apoptosis. However, at low UV doses the cleavage complex level was very low and the complexes were repaired. Surprisingly, we found that high levels of stable cleavage complexes were not only found in UV-irradiated cells but also in untreated cells that underwent apoptosis. A possible role of human topoisomerase I in apoptosis is discussed.