Structural characterization of bulk and nanoparticle lead halide perovskite thin films by (S)TEM techniques.

Lead halide (APbX3) perovskites, in polycrystalline thin films but also perovskite nanoparticles (NPs) has demonstrated excellent performance to implement a new generation of photovoltaic and photonic devices. The structural characterization of APbX3 thin films using (scanning) transmission electron microscopy ((S)TEM) techniques can provide valuable information that can be used to understand and model their optoelectronic performance and device properties. However, since APbX3 perovskites are soft materials, their characterization using (S)TEM is challenging. Here, we study and compare the structural properties of two different metal halide APbX3 perovskite thin films: bulk CH3NH3PbI3 prepared by spin-coating of the precursors in solution and CsPbBr3 colloidal NPs synthetized and deposited by doctor blading. Both specimen preparation methods and working conditions for analysis by (S)TEM are properly optimized. We show that CH3NH3PbI3 thin films grown by a one-step method are composed of independent grains with random orientations. The growth method results in the formation of tetragonal perovskite thin films with good adherence to an underlying TiO2 layer, which is characterized by a photoluminescence (PL) emission band centered at 775 nm. The perovskite thin films based on CsPbBr3 colloidal NPs, which are used as the building blocks of the film, are preserved by the deposition process, even if small gaps are observed between adjacent NPs. The crystal structure of CsPbBr3 NPs is cubic, which is beneficial for optical properties due to its optimal band gap. The absorption and PL spectra measured in both the thin film and the colloidal solution of CsPbBr3 NPs are very similar, indicating a good homogeneity of the thin films and the absence of aggregation of NPs. However, a particular care was required to avoid long electron irradiation times during our structural studies, even at a low voltage of 80 kV, as the material was observed to decompose through Pb segregation.

Analysis of biomass sugars using a novel HPLC method.

The precise quantitative analysis of biomass sugars is a very important step in the conversion of biomass feedstocks to fuels and chemicals. However, the most accurate method of biomass sugar analysis is based on the gas chromatography analysis of derivatized sugars either as alditol acetates or trimethylsilanes. The derivatization method is time consuming but the alternative high-performance liquid chromatography (HPLC) method cannot resolve most sugars found in biomass hydrolysates. We have demonstrated for the first time that by careful manipulation of the HPLC mobile phase, biomass monomeric sugars (arabinose, xylose, fructose, glucose, mannose, and galactose) can be analyzed quantitatively and there is excellent baseline resolution of all the sugars. This method was demonstrated for standard sugars, pretreated corn stover liquid and solid fractions. Our method can also be used to analyze dimeric sugars (cellobiose and sucrose).

Identification of microbial inhibitory functional groups in corn stover hydrolysate by carbon-13 nuclear magnetic resonance spectroscopy.

Dilute-acid biomass hydrolysates contain biomass degradation products that are inhibitory to cell growth and fermentation. Overliming with Ca(OH)2 has been found to be one of the most effective methods for detoxifying the dilute-acid hydrolysate for ethanol production. However, the mechanism of overliming is not well understood. Carbon-13 nuclear magnetic resonance (13C-NMR) spectroscopy was used to elucidate the functional groups involved in the overliming reaction. The 13C-NMR spectra showed that the major functional groups removed during the overliming process were aliphatic and aromatic acids or esters, and other aromatic and aliphatic compounds. Ketone and aldehyde functionalities were not detected in the spectra. This is the first time that 13C-NMR has been used to elucidate the overliming reaction.

Improved method of analysis of biomass sugars using high-performance liquid chromatography.

The precise quantitative analysis of biomass derived sugars is a very important step in the conversion of biomass feedstocks to fuels and chemicals. However, the most accurate method of biomass sugar analysis is based on the gas chromatography analysis of derivatized sugars either as alditol acetates or trimethylsilanes. The derivatization method is time-consuming but the alternative HPLC method cannot resolve most sugars found in biomass hydrolysates. We have demonstrated for the first time that by careful manipulation of the HPLC mobile phase, biomass monomeric sugars (arabinose, xylose, fructose, glucose, mannose, and galactose) can be analyzed quantitatively and there is excellent baseline resolution of all the sugars. This was demonstrated for both standard sugars and corn stover hydrolysates. Our method can also be used to analyze dimmeric sugars (cellobiose and sucrose).

Control elements of Dictyostelium discoideum prespore specific gene 3B.

Expression of the prespore-specific gene 3B in Dictyostelium discoideum Ax-2 cells is first detectable late in development with 3B mRNA levels peaking at 18 h (Corney et al., 1990). Sequence analysis of 3B cDNA and genomic clones revealed two exons, 319bp and 341bp long, separated by an 82bp intron, which encode a 219 residue protein with no significant similarity to any other reported gene product. Transcription starts at an A residue 45bp upstream from the translation initiation codon, preceded by a TATA-like sequence and an oligo-dT stretch. The 5' flanking sequence of the 3B gene is extremely A + T rich but contains five G/C rich stretches, each approximately 7bp long, which have strong sequence similarity to the G boxes found upstream of other developmentally regulated Dictyostelium genes. Analysis of both 3B promoter-CAT reporter gene and 3B promoter-lacZ reporter gene constructs showed that 908bp of 5' flanking sequence is sufficient to confer correct developmental and cell-type specific regulation. Sequential 5' deletion analysis revealed that positive elements lie upstream of position -304 and that negative element(s) lie between positions -264 and -241. Nevertheless, a 286bp promoter fragment containing only sequence located downstream of position -241 directed essentially correct reporter gene expression. Point mutation analysis identified cis-acting elements within this 'sufficient' promoter fragment which activate transcription (G box V and psp-AT type sequences). A short (56bp) region of the 3B promoter sequence containing both G box IV and the psp-AT type element binds two types of nuclear factor, one present in cells throughout development and a second that appears only in late development with a time course comparable to 3B gene induction.

Identification of novel elements which regulate the cell-type specificity of Dictyostelium 7E gene expression.

Previously, we have identified the Dictyostelium 7E gene promoter and shown that it is capable of driving expression in the same temporal and cAMP responsive manner as the endogenous gene during development. Furthermore, we have mapped the corresponding transcriptional regulatory sequences within the promoter. In the present study we used the lacZ reporter gene system to examine the role of 7E promoter elements in regulating cell-type specific expression during Dictyostelium morphogenesis. In situ detection of beta-galactosidase activity revealed that expression was induced within anterior prestalk cells at approximately 18 h of development. Subsequently, we found that promoter activity was independently regulated in subpopulations of prestalk cells. Element(s) upstream of position - 532 were necessary for expression in pstA cells while more proximal elements (located downstream of position - 426) were capable of directing expression in pstO cells. Deletion of a G-rich element ('GGT' box; 5'-GGT GAT GA-3') located between positions - 159 and - 152 resulted both in a loss of expression in pstA cells and aberrant expression in the prespore zone. Furthermore, the spatial organisation of reporter gene expression directed by this construct during culmination delineated a population of cells that have not been previously defined. These data suggest that the 7E gene is independently regulated in subpopulations of prestalk cells during development.

The carboxyl terminus of Dictyostelium discoideum protein 1I encodes a functional glycosyl-phosphatidylinositol signal sequence.

The 1I gene is expressed in the prespore cells of culminating Dictyostelium discoideum. The open reading frame of 1I cDNA encodes a protein of 155 amino acids with hydrophobic segments at both its NH(2)- and COOH-termini that are indicative of a glycosyl-phosphatidylinositol (GPI)-anchored protein. A hexaHis-tagged form of 1I expressed in D. discoideum cells appeared on Western blot analysis as a doublet of 27 and 24 kDa, with a minor polypeptide of 22 kDa. None of the polypeptides were released from the cell surface with bacterial phosphatidylinositol-specific phospholipase C, although all three were released upon nitrous acid treatment, indicating the presence of a phospholipase-resistant GPI anchor. Further evidence for the C-terminal sequence of 1I acting as a GPI attachment signal was obtained by replacing the GPI anchor signal sequence of porcine membrane dipeptidase with that from 1I. Two constructs of dipeptidase with the 1I GPI signal sequence were constructed, one of which included an additional six amino acids in the hydrophilic spacer. Both of the resultant constructs were targeted to the surface of COS cells and were GPI-anchored as shown by digestion with phospholipase C, indicating that the Dictyostelium GPI signal sequence is functional in mammalian cells. Site-specific antibodies recognising epitopes either side of the expected GPI anchor attachment site were used to determine the site of GPI anchor attachment in the constructs. These parallel approaches show that the C-terminal signal sequence of 1I can direct the addition of a GPI anchor.

Stereoselective and regioselective reaction of cyclic ortho esters with phenols.

Cyclic ortho esters undergo stereoselective and regioselective reaction with phenols when treated with BF(3) x OEt(2) at low temperatures. Attack of the phenol on the ortho ester occurs at an open carbon para to electron-donating groups on the phenol ("C-addition") or at the phenolic hydroxyl group ("O-addition") depending on the nature of the cation formed from reaction of the ortho ester and BF(3) x OEt(2). Products resulting from O-addition undergo reversion to a mixture of starting phenol, C-addition product, and O-addition product if treated with BF(3) x OEt(2) at room temperature, but C-addition products are stable under the same conditions. X-ray structural analysis of the C-addition compound indicates that its stereochemistry is opposite to that observed in reaction of similar ortho esters with chloride from TMSCl. However, the stereochemistry of the reaction can be rationalized by the ability of the ortho ester to isomerize via an intermediate benzylic cation and examination of the preferred trajectory of attack of the nucleophile on the intermediate oxonium ion.

In vitro degradation of natural insoluble lignin in aqueous media by the extracellular peroxidases of Phanerochaete chrysosporium.

The lignin peroxidases (LIP) and manganese peroxidases (MNP) of Phanerochaete chrysosporium catalyze a wide range of lignin depolymerization reactions with lignin models and synthetic lignins in solution. However, their ability to degrade insoluble natural lignin in aqueous media has not been demonstrated. Insoluble isolated poplar lignin similar to natural lignin was treated in vitro in aqueous media for 12 h with LIP, MNP, and both. Treatment with MNP alone slightly increased the solid mass and produced measurable amounts of lignin-derived 2,6-dimethoxyhydroquinone and 2-methoxyhydroquinone but did not appreciably decrease the total lignin content. Treatment with LIP alone did not decrease the mass but produced measurable amounts of lignin-derived p-hydroxybenzoic acid and slightly decreased the lignin content. Finally, treatment with LIP and MNP together decreased the solid mass by 11%, decreased the lignin content by 5%, and released low-concentration compounds with mass spectra containing the typical lignin-derived electron-impact fragments of mass 107, 137, 151, 167, and 181. These results suggest that MNP increases the effectiveness of LIP-mediated lignin degradation.

In vitro degradation of insoluble lignin in aqueous media by lignin peroxidase and manganese peroxidase.

The abilities of lignin peroxidase (LIP) and manganese peroxidase (MNP) from Phanerochaete chrysosporium to degrade an insoluble hardwood lignin in vitro in aqueous media were tested. Neither LIP nor MNP appreciably changed the mass or lignin content, although both produced small amounts of unique solubilized lignin fragments. Treatment with both LIP and MNP, however, decreased the mass by 11%, decreased the lignin content by 5.1% (4.2% as total weight), and solubilized unique lignin-derived molecules. These results suggest that LIP and MNP synergistically degrade high molecular weight insoluble lignin, but singly, neither enzyme is sufficient to effect lignin degradation.

Dosimetric parameters of a modified set of wedges for use with asymmetric fields of a 6 MV linear accelerator.

A set of standard wedge filters has been modified for use with half-collimated beams of a 6 MV linear accelerator. The position of the standard size wedge filter has been shifted as far to one side of the wedge plate to ensure optimum half-collimated field coverage (up to 20 x 30 cm) required in certain clinical situations. Dosimetric parameters were normalized at 1.5 cm depth and at an off-axis reference point (3.5 cm from the central axis of the collimator at 100 cm SSD. The shapes of the wedged profile and isodose curves of the modified wedges remained similar to those of standard wedges. Data presented include wedge transmission factors, wedge angles, beam profiles, and isodose distributions. The clinical advantages of using modified wedge filters (larger field size, larger transmission, and smaller weight) over standard large wedges is discussed.

A Xenopus borealis homeobox gene expressed preferentially in posterior ectoderm.

We have isolated a new homeobox-encoding gene (XhoxB.1) from Xenopus borealis which has a homeobox exon identical to that of the murine Hox1.7 gene, except for one amino acid. XhoxB.1 transcripts of 2.1 and 7.0 kb are first detected at the late gastrula stage, accumulate until the tailbud stage and are most abundant in the posterior third of the dorsal part of the embryo.

A developmentally regulated gene encodes the dictyostelium homolog of yeast ribosomal protein S4 and mammalian LLRep3 proteins.

We report the sequence and expression of a single-copy gene from Dictyostelium discoideum which encodes the homolog of yeast ribosomal protein S4, a protein located on the small ribosomal subunit and known to play an important role in maintaining translational fidelity. Over a highly conserved central region, the Dictyostelium protein has 78% sequence similarity to the yeast protein and 83% sequence similarity to mammalian S4 protein homologs, the LLRep3 proteins. The Dictyostelium gene encodes a polypeptide 28,717 Da in size and hence this ribosomal protein has been named rp29. The N-terminal sequence of the Dictyostelium rp29 protein is extended by 61 amino acids and 14 amino acids compared to the mammalian and yeast proteins, respectively, and the C-terminus is correspondingly 15 amino acids or 2 amino acids shorter. Although the coding region of the rp29 gene is present on a single exon, a 157bp intron interrupts the 5' untranslated region and unusually contains four direct repeats of the sequence TCAATCT. The gene is expressed maximally during vegetative growth but a second peak of expression also occurs late in development which is restricted to prestalk cells; rp29 is the first Dictyostelium ribosomal protein gene reported which shows prestalk-specific developmental expression. During each round of expression, only a single 0.9kb transcript is produced which is similar in size to the yeast S4 ribosomal protein transcript (0.8kb) but markedly smaller than the mammalian LLRep3 mRNA (1.7kb) due to a much shorter 5' untranslated region.

Sequence and expression analysis of a cAMP-responsive gene regulated during late development of Dictyostelium discoideum.

The 7E gene is expressed late in normal development of Dictyostelium discoideum after pseudoplasmodium formation. After disaggregation of the developing cells, transcription of this gene depends entirely on exogenous 3'5' cyclic AMP (cAMP). The 5' flanking region of the 7E gene contains two TATA box-oligo (dT) promoter motifs but analysis of 7E gene expression by primer extension shows only a single primary transcript with transcription initiating immediately after the most proximal promoter motif during development or in disaggregated cells in the presence of exogenous cAMP. Four C-rich sequences lie within 350bp upstream of the cap site, analogous to the upstream elements implicated in the cAMP regulation of several other Dictyostelium genes expressed in development.

Cell-type-specific genes expressed late in Dictyostelium development show markedly different responses to 3'5' cyclic AMP.

The maintenance of late gene expression by 3'5' cyclic AMP was re-examined using several newly isolated cell-type-specific genes. Expression of all the prespore-enriched genes ceased immediately upon disaggregation of developing cells and pre-existing mRNA was rapidly degraded. For most genes, cAMP had little or no effect either alone or in combination with conditioned medium factors. The expression of the non-cell-type-specific genes 7E and 2C also ceased upon cell disaggregation but cAMP triggered a full re-induction of expression although the timing of the response differed markedly between these two genes. In contrast to earlier interpretations, these data argue that for none of these late prespore genes is cAMP alone sufficient for the maintenance of expression. The responses of the two prestalk mRNAs examined were gene-specific. Prestalk 5D mRNA decayed slowly upon disaggregation and was partially stabilized by cAMP whereas prestalk 5G mRNA increased upon disaggregation and was inhibited by cAMP.

Developmental expression and characterization of the gene encoding spore coat protein SP60 in Dictyostelium discoideum.

The complete coding sequence, upstream sequence and developmental expression of Dictyostelium discoideum AX2 spore coat protein gene SP60 is reported. The gene contains two exons, 154bp and 1121bp long, separated by a 119bp intron, and encodes a protein of 46,925 molecular weight plus a 23-amino-acid hydrophobic leader sequence. The N-terminus of the mature protein consists of four copies of a perfect hexapeptide repeat (GDWNNN). The central region is rich in cysteine residues, including four highly conserved cysteine-rich repeats with homology to 'EGF-like' repeats. The C-terminus is aspartate-rich and composed of multiple imperfect copies of a D(G/D)DYD repeat followed by several repeats of the tetrapeptide DNDW and derived sequences. A TATA box promoter motif juxtaposed to an oligo(dA) stretch lies 52bp upstream of the main transcriptional start site of the gene. Six AC-rich boxes occur in the region -327 to -556, all of which contain the consensus sequence CACAC. Two GC-rich boxes and a C-rich element (TTACCCCA) are also present upstream. Another open reading frame is positioned a short distance downstream of the SP60 gene in the opposite transcriptional orientation. Expression of the SP60 gene ceases upon disaggregation to single cells and cannot be restored by high levels of extracellular cAMP either alone or in combination with conditioned medium factors.

Developmental regulation of cell-type-enriched mRNAs in Dictyostelium discoideum.

We describe sixteen new families of cDNA clones representing mRNAs that are expressed preferentially in either prespore or prestalk cells during development of Dictyostelium discoideum and two new mRNAs that are expressed in a non-cell-type-specific manner. None of the prespore-enriched mRNAs are detectable in Dictyostelium cells until 13-15 h of development but then they increase dramatically and peak at 18-22 h. Upon dissociation of developing aggregates, all these mRNAs rapidly decay to low levels. In marked contrast to data presented for prespore genes by other workers, cyclic AMP either has no effect on the mRNA levels in dissociated cells or is only weakly effective in restoring normal expression. A prestalk-enriched mRNA examined, 5G mRNA, is similarly expressed late in development but is also expressed in vegetative cells. The level of 5G mRNA is only moderately affected by cell disaggregation.

Two cyclic AMP-regulated genes from Dictyostelium discoideum encode homologous proteins.

Expression of the 7E and 2C genes late in Dictyostelium development ceases upon cell disaggregation but, in contrast to many other genes we have studied, expression is fully restored by exogenous cAMP (A. J. Richards et al., submitted). The 7E and 2C genes encode polypeptides of similar size (9220 and 10573 Daltons, respectively), each of which contains an unusually high proportion of serine plus glycine residues (41% and 59%, respectively). Each protein possesses a relatively serine-rich N-terminus and glycine-rich C-terminus and contains the conserved sequence S(X)SSS(X2)SS(X)SS(X2)SFGS. These data suggest that genes 7E and 2C may have arisen by duplication of a common ancestor. Computer analysis indicates that both gene products are probably intracellular structural proteins that form extended coil structures.

Analysis of specific mRNA destabilization during Dictyostelium development.

A number of specific mRNAs are destabilized upon disaggregation of developing Dictyostelium discoideum cells. Analysis of a family of cloned genes indicates that only prespore-enriched mRNAs are affected; constitutive mRNAs that are expressed throughout development and mRNAs that accumulate preferentially in prestalk cells are stable under these conditions. The decay of sensitive prespore mRNAs can be halted by allowing the cells to reaggregate, indicating that destabilization occurs by the progressive selection of individual molecules rather than on all members of an mRNA subpopulation at the time of disaggregation. Individual molecules of the sensitive mRNA species remain engaged in protein synthesis in the disaggregated cells until selected. Destabilization of sensitive mRNAs is induced by cell dissociation even in the presence of concentrations of nogalamycin that inhibit RNA synthesis. The reported prevention of disaggregation-induced mRNA decay by actinomycin D and daunomycin is therefore probably a secondary effect unrelated to the inhibition of transcription.