Distinctive interactions of the Arabidopsis homolog of the 30 kD subunit of the cleavage and polyadenylation specificity factor (AtCPSF30) with other polyadenylation factor subunits

BACKGROUND: The Arabidopsis ortholog of the 30 kD subunit of the mammalian Cleavage and Polyadenylation Specificity Factor (AtCPSF30) is an RNA-binding endonuclease that is associated with other Arabidopsis CPSF subunits (orthologs of the 160, 100, and 73 kD subunits of CPSF). In order to further explore the functions of AtCPSF30, the subcellular distribution of the protein was examined by over-expressing fusion proteins containing fluorescent reporters linked to different CPSF subunits. RESULTS: It was found that AtCPSF30 by itself localizes, not to the nucleus, but to the cytoplasm. AtCPSF30 could be found in the nucleus when co-expressed with AtCPSF160 or AtCPSF73(I), one of the two Arabidopsis orthologs of CPSF73. This re-directing of AtCPSF30 indicates that AtCPSF30 is retained in the nucleus via interactions with either or both of these other CPSF subunits. Co-expression of AtCSPF30 with AtCPSF100 altered the location, not of AtCPSF30, but rather of AtCPSF100, with these proteins residing in the cytoplasm. Deletion of plant-specific N- or C-terminal domains of AtCPSF30 abolished various of the interactions between AtCPSF30 and other CPSF subunits, suggesting that the plant CPSF complex assembles via novel protein-protein interactions. CONCLUSION: These results suggest that the nuclear CPSF complex in plants is a dynamic one, and that the interactions between AtCPSF30 and other CPSF subunits are different from those existing in other eukaryotes

Fungal Desaturases and Related Methods

The presently-disclosed subject matter provides isolated nucleic acid and amino acid sequences encoding mushroom desaturase polypeptides that are active with both palmitic and stearic acid, as well as vectors and transgenic plant cells comprising nucleic acids of the presently-disclosed subject matter. The presently-disclosed subject matter further provides methods of producing monounsaturated fatty acids, such as palmitoleic acid (16:1), and monounsaturated fatty acids prepared by the methods disclosed herein

SOLUBILITY AND DISSOLUTION RATE ENHANCEMENT OF EZETIMIBE BY SOLID DISPERSION AND PELLETIZATION TECHNIQUES USING SOLUPLUS AS CARRIER

Objective: In the present investigation, an attempt was made to improve the surface characters and solubility of the drug by solid dispersion and coating it on the nonpareil sugar beads as pellets. Methods: Ezetimibe solid dispersions were prepared by kneading method using soluplus. Crospovidone was added as a disintegrant in pellets. Ezetimibe pellets were prepared by dissolving soluplus and crospovidone in ethanol in different ratios and coated on nonpareil sugar beads as a drug layer by pan coating technique. Various physicochemical parameters like particle size, friability, angle of repose and drug content were evaluated for the prepared solid dispersions and pellet formulations. In vitro dissolution studies were carried out in 1% SLS using USP apparatus II. FTIR and SEM analysis were performed for solid dispersions, pellet formulations and its polymers to determine the interactions and surface characteristics. Results: The physicochemical parameters were within the specified I. P limits. It was observed that the solid dispersion formulation ED5 showed better dissolution rate to the extent of 1.07 folds and 1.95 folds when compared to a marketed formulation and the pure drug, respectively. Similarly, pellet formulation EP5 containing 1:5 ratio of ezetimibe to soluplus showed an improved dissolution rate to the extent of 1.173 folds and 2.136 folds when compared to the marketed formulation and the pure drug, respectively. FTIR analysis revealed that there was no major interaction between the drug and the excipients.  Conclusion: From the present study, it was observed that the solubility of ezetimibe was enhanced by soluplus in pellet formulations when compared to solid dispersions

SOLUBILITY AND DISSOLUTION RATE ENHANCEMENT OF EZETIMIBE BY SOLID DISPERSION AND PELLETIZATION TECHNIQUES

Objective: In the present investigation, an attempt was made to improve the surface characters and solubility of the drug by solid dispersion and coating it on the non-pareil sugar beads as pellets. Methods: Ezetimibe solid dispersions were prepared by solvent evaporation technique using Kollidon VA64 as binder and solubility enhancer. Crospovidone as disintegrant and ethanol was used as solvent. Ezetimibe pellets were prepared by dissolving ezetimibe, kollidonVA64, and crospovidone in ethanol in different ratios and coated on non-pareil sugar beads as a drug layer by pan coating technique. Results: All the formulations were further evaluated for physicochemical parameters such as particle size, friability, angle of repose, and drug content. In vitro dissolution studies were carried out in 1% sodium lauryl sulfate using USP apparatus II. Conclusion: It was observed that the dissolution rate of the solid dispersion formulation ESD5 showed better dissolution rate to the extent of 1.05 folds and 1.824 folds when compared to a marketed formulation and pure drug, respectively. Similarly, formulation EPL5 containing 1:5 ratio of ezetimibe to Kollidon VA64 showed improved dissolution rate to the extent of 1.091 folds and 1.986 folds when compared to the marketed formulation and pure drug, respectively. Majority of the formulations displayed first-order release kinetics and were found to be linear with R2 values in the range of 0.874–0.993. Fourier transform infrared analysis revealed that there was no major interaction between the drug and excipients used in the design of formulation. Scanning electron microscopy analysis was performed for solid dispersions, pellet formulations, and its polymers to determine the surface characteristics

The clinical behavior of mixed ductal/lobular carcinoma of the breast: a clinicopathologic analysis

<p>Abstract</p> <p>Background</p> <p>To date, the clinical presentation and prognosis of mixed ductal/lobular mammary carcinomas has not been well studied, and little is known about the outcome of this entity. Thus, best management practices remain undetermined due to a dearth of knowledge on this topic.</p> <p>Methods</p> <p>In this paper, we present a clinicopathologic analysis of patients at our institution with this entity and compare them to age-matched controls with purely invasive ductal carcinoma (IDC) and historical data from patients with purely lobular carcinoma and also stain-available tumor specimens for E-cadherin. We have obtained 100 cases of ductal and 50 cases of mixed ductal/lobular breast carcinoma.</p> <p>Results</p> <p>Clinically, the behavior of mixed ductal/lobular tumors seemed to demonstrate some important differences from their ductal counterparts, particularly a lower rate of metastatic spread but with a much higher rate of second primary breast cancers.</p> <p>Conclusions</p> <p>Our data suggests that mixed ductal/lobular carcinomas are a distinct clinicopathologic entity incorporating some features of both lobular and ductal carcinomas and representing a pleomorphic variant of IDC.</p

SOLUBILITY AND DISSOLUTION RATE ENHANCEMENT OF TELMISARTAN BY SOLID DISPERSION AND PELLETIZATION TECHNIQUES USING SOLUPLUS AS CARRIER

Objective: In the present investigation, an attempt was made to improve the surface characters and solubility of the drug by solid dispersion and coating it on the nonpareil sugar beads as pellets. Methods: Telmisartan solid dispersions were prepared by kneading method using soluplus. Crospovidone was added as disintegrant in pellets. Telmisartan pellets were prepared by dissolving soluplus and crospovidone in ethanol in different ratios and coated on nonpareil sugar beads as a drug layer by pan coating technique. Various physicochemical parameters like particle size, friability, angle of repose and drug content were evaluated for the prepared solid dispersions and pellet formulations. In vitro dissolution studies were carried out in pH 7.5 phosphate buffer using USP apparatus II. Fourier Transform Infrared Spectrometry, Differential Scanning Calorimetry and Scanning Electron Microscopic analysis were performed for solid dispersions, pellet formulations and its polymers to determine the interactions and surface characteristics. Results: The physicochemical parameters were within the specified I. P limits. It was observed that the solid dispersion formulation TS5 containing&nbsp;1:5 ratio of telmisartan to soluplus&nbsp;showed&nbsp;better dissolution rate to the extent of 1.143 folds and 2.033 folds when compared to a marketed formulation and the pure drug, respectively. Similarly, pellet formulation TP3&nbsp;containing&nbsp;1:3 ratio of telmisartan to soluplus showed an improved dissolution rate to the extent of 1.221 folds and 2.170 folds when compared to the marketed formulation and the pure drug, respectively. FTIR and DSC analysis revealed that there was no major interaction between the drug and the&nbsp;excipients.&nbsp; Conclusion: From the present study, it was observed that the solubility of telmisartan was enhanced by soluplus in pellet formulations when compared to solid dispersions

Non-antibiotic selection systems for soybean somatic embryos: the lysine analog aminoethyl-cysteine as a selection agent

<p>Abstract</p> <p>Background</p> <p>In soybean somatic embryo transformation, the standard selection agent currently used is hygromycin. It may be preferable to avoid use of antibiotic resistance genes in foods. The objective of these experiments was to develop a selection system for producing transgenic soybean somatic embryos without the use of antibiotics such as hygromycin.</p> <p>Results</p> <p>When tested against different alternate selection agents our studies show that 0.16 μg/mL glufosinate, 40 mg/L isopropylamine-glyphosate, 0.5 mg/mL (S-(2 aminoethyl)-L-cysteine) (AEC) and the acetolactate synthase (ALS) inhibitors Exceed^®and Synchrony^®both at 150 μg/mL inhibited soybean somatic embryo growth. Even at the concentration of 2 mg/mL, lysine+threonine (LT) were poor selection agents. The use of AEC may be preferable since it is a natural compound. Unlike the plant enzyme, dihydrodipicolinate synthase (DHPS) from <it>E. coli </it>is not feed-back inhibited by physiological concentrations of lysine. The <it>dapA </it>gene which codes for <it>E. coli </it>DHPS was expressed in soybean somatic embryos under the control of the CaMV 35S promoter. Following introduction of the construct into embryogenic tissue of soybean, transgenic events were recovered by incubating the tissue in liquid medium containing AEC at a concentration of 5 mM. Only transgenic soybeans were able to grow at this concentration of AEC; no escapes were observed.</p> <p>Conclusion</p> <p>Genetically engineered soybeans expressing a lysine insensitive DHPS gene can be selected with the non-antibiotic selection agent AEC. We also report here the inhibitory effects of glufosinate, (isopropylamine-glyphosate) (Roundup^®), AEC and the ALS inhibitors Exceed^®and Synchrony^®against different tissues of soybean</p

Arabidopsis mRNA polyadenylation machinery: comprehensive analysis of protein-protein interactions and gene expression profiling

BACKGROUND: The polyadenylation of mRNA is one of the critical processing steps during expression of almost all eukaryotic genes. It is tightly integrated with transcription, particularly its termination, as well as other RNA processing events, i.e. capping and splicing. The poly(A) tail protects the mRNA from unregulated degradation, and it is required for nuclear export and translation initiation. In recent years, it has been demonstrated that the polyadenylation process is also involved in the regulation of gene expression. The polyadenylation process requires two components, the cis-elements on the mRNA and a group of protein factors that recognize the cis-elements and produce the poly(A) tail. Here we report a comprehensive pairwise protein-protein interaction mapping and gene expression profiling of the mRNA polyadenylation protein machinery in Arabidopsis. RESULTS: By protein sequence homology search using human and yeast polyadenylation factors, we identified 28 proteins that may be components of Arabidopsis polyadenylation machinery. To elucidate the protein network and their functions, we first tested their protein-protein interaction profiles. Out of 320 pair-wise protein-protein interaction assays done using the yeast two-hybrid system, 56 (approximately 17%) showed positive interactions. 15 of these interactions were further tested, and all were confirmed by co-immunoprecipitation and/or in vitro co-purification. These interactions organize into three distinct hubs involving the Arabidopsis polyadenylation factors. These hubs are centered around AtCPSF100, AtCLPS, and AtFIPS. The first two are similar to complexes seen in mammals, while the third one stands out as unique to plants. When comparing the gene expression profiles extracted from publicly available microarray datasets, some of the polyadenylation related genes showed tissue-specific expression, suggestive of potential different polyadenylation complex configurations. CONCLUSION: An extensive protein network was revealed for plant polyadenylation machinery, in which all predicted proteins were found to be connecting to the complex. The gene expression profiles are indicative that specialized sub-complexes may be formed to carry out targeted processing of mRNA in different developmental stages and tissue types. These results offer a roadmap for further functional characterizations of the protein factors, and for building models when testing the genetic contributions of these genes in plant growth and development

A Polyadenylation Factor Subunit Implicated in Regulating Oxidative Signaling in Arabidopsis thaliana

BACKGROUND: Plants respond to many unfavorable environmental conditions via signaling mediated by altered levels of various reactive oxygen species (ROS). To gain additional insight into oxidative signaling responses, Arabidopsis mutants that exhibited tolerance to oxidative stress were isolated. We describe herein the isolation and characterization of one such mutant, oxt6. METHODOLOGY/PRINCIPAL FINDINGS: The oxt6 mutation is due to the disruption of a complex gene (At1g30460) that encodes the Arabidopsis ortholog of the 30-kD subunit of the cleavage and polyadenylation specificity factor (CPSF30) as well as a larger, related 65-kD protein. Expression of mRNAs encoding Arabidopsis CPSF30 alone was able to restore wild-type growth and stress susceptibility to the oxt6 mutant. Transcriptional profiling and single gene expression studies show elevated constitutive expression of a subset of genes that encode proteins containing thioredoxin- and glutaredoxin-related domains in the oxt6 mutant, suggesting that stress can be ameliorated by these gene classes. Bulk poly(A) tail length was not seemingly affected in the oxt6 mutant, but poly(A) site selection was different, indicating a subtle effect on polyadenylation in the mutant. CONCLUSIONS/SIGNIFICANCE: These results implicate the Arabidopsis CPSF30 protein in the posttranscriptional control of the responses of plants to stress, and in particular to the expression of a set of genes that suffices to confer tolerance to oxidative stress

Performance of the CMS High Granularity Calorimeter prototype to charged pion beams of 20−-300 GeV/c

The upgrade of the CMS experiment for the high luminosity operation of the LHC comprises the replacement of the current endcap calorimeter by a high granularity sampling calorimeter (HGCAL). The electromagnetic section of the HGCAL is based on silicon sensors interspersed between lead and copper (or copper tungsten) absorbers. The hadronic section uses layers of stainless steel as an absorbing medium and silicon sensors as an active medium in the regions of high radiation exposure, and scintillator tiles directly readout by silicon photomultipliers in the remaining regions. As part of the development of the detector and its readout electronic components, a section of a silicon-based HGCAL prototype detector along with a section of the CALICE AHCAL prototype was exposed to muons, electrons and charged pions in beam test experiments at the H2 beamline at the CERN SPS in October 2018. The AHCAL uses the same technology as foreseen for the HGCAL but with much finer longitudinal segmentation. The performance of the calorimeters in terms of energy response and resolution, longitudinal and transverse shower profiles is studied using negatively charged pions, and is compared to GEANT4 predictions. This is the first report summarizing results of hadronic showers measured by the HGCAL prototype using beam test data.Comment: To be submitted to JINS