cis-Golgi phosphate transporters harboring an EXS domain are essential for plant growth and development.

Cell wall synthesis and protein glycosylation require the import of nucleotide diphosphate-sugar conjugates into the Golgi that must be counterbalanced by phosphate (Pi) export. Numerous Golgi nucleotide-sugar transporters have been characterized, but transporters mediating Golgi Pi export remain poorly understood. We used plant and yeast genetics to characterize the role of 2 Arabidopsis (Arabidopsis thaliana) proteins possessing an EXS domain, namely ERD1A and ERD1B, in Golgi Pi homeostasis. ERD1A and ERD1B localized in cis-Golgi and were broadly expressed in vegetative and reproductive tissues. We identified ERD1 putative orthologs in algae, bryophytes, and vascular plants. Expressing ERD1A and ERD1B in yeast complemented the erd1 mutant phenotype of cellular Pi loss via exocytosis associated with reduced Golgi Pi export. The Arabidopsis erd1a mutant had a similar phenotype of apoplastic Pi loss dependent on exocytosis. ERD1A overexpression in Nicotiana benthamiana and Arabidopsis led to partial mislocalization of ERD1A to the plasma membrane and specific Pi export to the apoplastic space. Arabidopsis erd1a had defects in cell wall biosynthesis, which were associated with reduced shoot development, hypocotyl growth, cell wall extensibility, root elongation, pollen germination, pollen tube elongation, and fertility. We identified ERD1 proteins as Golgi Pi exporters that are essential for optimal plant growth and fertility.

Endoplasmic reticulum calnexins participate in the primary root growth response to phosphate deficiency.

Accumulation of incompletely folded proteins in the endoplasmic reticulum (ER) leads to ER stress, activates ER protein degradation pathways, and upregulates genes involved in protein folding. This process is known as the unfolded protein response (UPR). The role of ER protein folding in plant responses to nutrient deficiencies is unclear. We analyzed Arabidopsis (Arabidopsis thaliana) mutants affected in ER protein quality control and established that both CALNEXIN (CNX) genes function in the primary root response to phosphate (Pi) deficiency. CNX1 and CNX2 are homologous ER lectins promoting protein folding of N-glycosylated proteins via the recognition of the GlcMan9GlcNAc2 glycan. Growth of cnx1-1 and cnx2-2 single mutants was similar to that of the wild type under high and low Pi conditions, but the cnx1-1 cnx2-2 double mutant showed decreased primary root growth under low Pi conditions due to reduced meristematic cell division. This phenotype was specific to Pi deficiency; the double mutant responded normally to osmotic and salt stress. Expression of CNX2 mutated in amino acids involved in binding the GlcMan9GlcNAc2 glycan failed to complement the cnx1-1 cnx2-2 mutant. The root growth phenotype was Fe-dependent and was associated with root apoplastic Fe accumulation. Two genes involved in Fe-dependent inhibition of primary root growth under Pi deficiency, the ferroxidase LOW PHOSPHATE 1 (LPR1) and P5-type ATPase PLEIOTROPIC DRUG RESISTANCE 2 (PDR2) were epistatic to CNX1/CNX2. Overexpressing PDR2 failed to complement the cnx1-1 cnx2-2 root phenotype. The cnx1-1 cnx2-2 mutant showed no evidence of UPR activation, indicating a limited effect on ER protein folding. CNX might process a set of N-glycosylated proteins specifically involved in the response to Pi deficiency.

The transcription and export complex THO/TREX contributes to transcription termination in plants.

Transcription termination has important regulatory functions, impacting mRNA stability, localization and translation potential. Failure to appropriately terminate transcription can also lead to read-through transcription and the synthesis of antisense RNAs which can have profound impact on gene expression. The Transcription-Export (THO/TREX) protein complex plays an important role in coupling transcription with splicing and export of mRNA. However, little is known about the role of the THO/TREX complex in the control of transcription termination. In this work, we show that two proteins of the THO/TREX complex, namely TREX COMPONENT 1 (TEX1 or THO3) and HYPER RECOMBINATION1 (HPR1 or THO1) contribute to the correct transcription termination at several loci in Arabidopsis thaliana. We first demonstrate this by showing defective termination in tex1 and hpr1 mutants at the nopaline synthase (NOS) terminator present in a T-DNA inserted between exon 1 and 3 of the PHO1 locus in the pho1-7 mutant. Read-through transcription beyond the NOS terminator and splicing-out of the T-DNA resulted in the generation of a near full-length PHO1 mRNA (minus exon 2) in the tex1 pho1-7 and hpr1 pho1-7 double mutants, with enhanced production of a truncated PHO1 protein that retained phosphate export activity. Consequently, the strong reduction of shoot growth associated with the severe phosphate deficiency of the pho1-7 mutant was alleviated in the tex1 pho1-7 and hpr1 pho1-7 double mutants. Additionally, we show that RNA termination defects in tex1 and hpr1 mutants leads to 3'UTR extensions in several endogenous genes. These results demonstrate that THO/TREX complex contributes to the regulation of transcription termination.

At14a-Like1 participates in membrane-associated mechanisms promoting growth during drought in Arabidopsis thaliana.

Limited knowledge of how plants regulate their growth and metabolism in response to drought and reduced soil water potential has impeded efforts to improve stress tolerance. Increased expression of the membrane-associated protein At14a-like1 (AFL1) led to increased growth and accumulation of the osmoprotective solute proline without negative effects on unstressed plants. Conversely, inducible RNA-interference suppression of AFL1 decreased growth and proline accumulation during low water potential while having no effect on unstressed plants. AFL1 overexpression lines had reduced expression of many stress-responsive genes, suggesting AFL1 may promote growth in part by suppression of negative regulatory genes. AFL1 interacted with the endomembrane proteins protein disulfide isomerase 5 (PDI5) and NAI2, with the PDI5 interaction being particularly increased by stress. PDI5 and NAI2 are negative regulatory factors, as pdi5, nai2, and pdi5-2nai2-3 mutants had increased growth and proline accumulation at low water potential. AFL1 also interacted with Adaptor protein2-2A (AP2-2A), which is part of a complex that recruits cargo proteins and promotes assembly of clathrin-coated vesicles. AFL1 colocalization with clathrin light chain along the plasma membrane, together with predictions of AFL1 structure, were consistent with a role in vesicle formation or trafficking. Fractionation experiments indicated that AFL1 is a peripheral membrane protein associated with both plasma membrane and endomembranes. These data identify classes of proteins (AFL1, PDI5, and NAI2) not previously known to be involved in drought signaling. AFL1-predicted structure, protein interactions, and localization all indicate its involvement in previously uncharacterized membrane-associated drought sensing or signaling mechanisms.

Once cleaved C-C bond was reformed: reversible C-C bond cleavage of dihydroindenyltitanium complexes.

The once cleaved carbon-carbon bond of the Cp moiety in 2 was recombined in indene products. Aslo, we propose a novel mechanism for the cleavage of the carbon-carbon bond of the Cp moiety.

Efficient purification of transglutaminase from recombinant Streptomyces platensis at various scales.

An efficient system for the fast and efficient purification of transglutaminase from recombinant Streptomyces platensis and expressed in Streptomyces lividans 25-2 is described. Because the purification procedure of this system is flexible, culture broth from laboratory (20 l) and pilot-plant (130 l) fermentations were used to purify the enzyme to electrophoretic homogeneity with high purity (90-95%) and yield (61-77%) within 1 or 2 days.

Numerical study of the effect of ventilation pattern on coarse, fine, and very fine particulate matter removal in partitioned indoor environment.

An indoor size-dependent particulate matter (PM) transport approach is developed to investigate coarse PM (PM10), fine PM (PM2.5), and very fine PM (PM1) removal behaviors in a ventilated partitioned indoor environment. The approach adopts the Eulerian large eddy simulation of turbulent flow and the Lagrangian particle trajectory tracking to solve the continuous airflow phase and the discrete particle phase, respectively. Model verification, including sensitivity tests of grid resolution and particle numbers, is conducted by comparison with the full-size experiments conducted previously. Good agreement with the measured mass concentrations is found. Numerical scenario simulations of the effect of ventilation patterns on PM removal are performed by using three common ventilation patterns (piston displacement, mixing, and cross-flow displacement ventilation) with a measured indoor PM10 profile in the Taipei metropolis as the initial condition. The temporal variations of suspended PM10, PM2.5, and PM1 mass concentrations and particle removal mechanisms are discussed. The simulated results show that for all the of the three ventilation patterns, PM2.5 and PM1 are much more difficult to remove than PM10. From the purpose of health protection for indoor occupants, it is not enough to only use the PM10 level as the indoor PM index. Indoor PM2.5 and PM1 levels should be also considered. Cross-flow displacement ventilation is more effective to remove all PM10, PM2.5, and PM1 than the other ventilation patterns. Displacement ventilation would result in more escaped particles and less deposited particles than mixing ventilation.

Rebuilding DEMATEL threshold value: an example of a food and beverage information system.

This study demonstrates how a decision-making trial and evaluation laboratory (DEMATEL) threshold value can be quickly and reasonably determined in the process of combining DEMATEL and decomposed theory of planned behavior (DTPB) models. Models are combined to identify the key factors of a complex problem. This paper presents a case study of a food and beverage information system as an example. The analysis of the example indicates that, given direct and indirect relationships among variables, if a traditional DTPB model only simulates the effects of the variables without considering that the variables will affect the original cause-and-effect relationships among the variables, then the original DTPB model variables cannot represent a complete relationship. For the food and beverage example, a DEMATEL method was employed to reconstruct a DTPB model and, more importantly, to calculate reasonable DEMATEL threshold value for determining additional relationships of variables in the original DTPB model. This study is method-oriented, and the depth of investigation into any individual case is limited. Therefore, the methods proposed in various fields of study should ideally be used to identify deeper and more practical implications.

An empirical research on customer satisfaction study: a consideration of different levels of performance.

Customer satisfaction is the key factor for successful and depends highly on the behaviors of frontline service providers. Customers should be managed as assets, and that customers vary in their needs, preferences, and buying behavior. This study applied the Taiwan Customer Satisfaction Index model to a tourism factory to analyze customer satisfaction and loyalty. We surveyed 242 customers served by one tourism factory organizations in Taiwan. A partial least squares was performed to analyze and test the theoretical model. The results show that perceived quality had the greatest influence on the customer satisfaction for satisfied and dissatisfied customers. In addition, in terms of customer loyalty, the customer satisfaction is more important than image for satisfied and dissatisfied customers. The contribution of this paper is to propose two satisfaction levels of CSI models for analyzing customer satisfaction and loyalty, thereby helping tourism factory managers improve customer satisfaction effectively. Compared with traditional techniques, we believe that our method is more appropriate for making decisions about allocating resources and for assisting managers in establishing appropriate priorities in customer satisfaction management.

G206D Mutation of Presenilin-1 Reduces Pen2 Interaction, Increases Aß42/Aß40 Ratio and Elevates ER Ca(2+) Accumulation.

Early-onset familial Alzheimer's disease (AD) is most commonly associated with the mutations in presenilin-1 (PS1). PS1 is the catalytic component of the γ-secretase complex, which cleaves amyloid precursor protein to produce amyloid-ß (Aß), the major cause of AD. Presenilin enhancer 2 (Pen2) is critical for activating γ-secretase and exporting PS1 from endoplasmic reticulum (ER). Among all the familial AD-linked PS1 mutations, mutations at the G206 amino acid are the most adjacent position to the Pen2 binding site. Here, we characterized the effect of a familial AD-linked PS1 G206D mutation on the PS1-Pen2 interaction and the accompanied alteration in γ-secretase-dependent and -independent functions. We found that the G206D mutation reduced PS1-Pen2 interaction, but did not abolish γ-secretase formation and PS1 endoproteolysis. For γ-secretase-dependent function, the G206D mutation increased Aß42 production but not Notch cleavage. For γ-secretase-independent function, this mutation disrupted the ER calcium homeostasis but not lysosomal calcium homeostasis and autophagosome maturation. Impaired ER calcium homeostasis may due to the reduced mutant PS1 level in the ER. Although this mutation did not alter the cell survival under stress, both increased Aß42 ratio and disturbed ER calcium regulation could be the mechanisms underlying the pathogenesis of the familial AD-linked PS1 G206D mutation.

Amyloid-beta (Aß) D7H mutation increases oligomeric Aß42 and alters properties of Aß-zinc/copper assemblies.

Amyloid precursor protein (APP) mutations associated with familial Alzheimer's disease (AD) usually lead to increases in amyloid ß-protein (Aß) levels or aggregation. Here, we identified a novel APP mutation, located within the Aß sequence (Aß(D7H)), in a Taiwanese family with early onset AD and explored the pathogenicity of this mutation. Cellular and biochemical analysis reveal that this mutation increased Aß production, Aß42/40 ratio and prolonged Aß42 oligomer state with higher neurotoxicity. Because the D7H mutant Aß has an additional metal ion-coordinating residue, histidine, we speculate that this mutation may promote susceptibility of Aß to ion. When co-incubated with Zn(2+) or Cu(2+), Aß(D7H) aggregated into low molecular weight oligomers. Together, the D7H mutation could contribute to AD pathology through a "double punch" effect on elevating both Aß production and oligomerization. Although the pathogenic nature of this mutation needs further confirmation, our findings suggest that the Aß N-terminal region potentially modulates APP processing and Aß aggregation, and further provides a genetic indication of the importance of Zn(2+) and Cu(2+) in the etiology of AD.

Characterization and large-scale production of recombinant Streptoverticillium platensis transglutaminase.

Recombinant Streptomyces platensis transglutaminase (MtgA) produced by the Streptomyces lividans transformant 25-2 was purified by ammonium sulfate fractionation, followed by CM-Sepharose CL-6B fast flow, and blue-Sepharose fast flow chromatography. The purification factor was approximately 33.2-fold, and the yield was 65%. The molecular weight of the purified recombinant MtgA was 40.0 KDa as estimated by SDS-PAGE. The optimal pH and the temperature for the enzyme activity were 6.0 and 55 degrees C, respectively, and the enzyme was stable at pH 5.0-6.0 and at temperature 45-55 degrees C. Enzyme activity was not affected by Ca(2+), Li(+), Mn(2+), Na(+), Fe(3+), K(+), Mg(2+), Al(3+), Ba(2+), Co(2+), EDTA, or IAA but was inhibited by Fe(2+), Pb(2+), Zn(2+), Cu(2+), Hg(2+), PCMB, NEM, and PMSF. Optimization of the fermentation medium resulted in a twofold increase of recombinant MtgA activity in both flasks (5.78 U/ml) and 5-l fermenters (5.39 U/ml). Large-scale productions of the recombinant MtgA in a 30-l air-lift fermenter and a 250-l stirred-tank fermenter were fulfilled with maximal activities of 5.36 and 2.54 U/ml, respectively.

Surgical treatment of midclavicular fractures: a prospective comparison of Knowles pinning and plate fixation.

Although several surgical techniques for midclavicular fractures have been reported, Knowles pinning has rarely been compared with plating. The purpose of this study is to compare the clinical results of these two alternative techniques. There were 88 patients with midclavicular fractures surgically treated with either a Knowles pin or a plate. All patients were followed up for 12 months with a shoulder score evaluation. The Knowles pin group included 56 patients, with an average age of 40.1 years. The plate group included 32 patients, with an average age of 38.2 years. Both groups were similar in injury mechanism and fracture types (all p values>0.5). Plating has a significantly longer operation time, larger wound incision, higher pain level, more analgesic use, more complications and more symptomatic hardware (all p value<0.05). The shoulder score, union rate and healing time are not significantly different between the two groups (all p values>0.2). In conclusion, if the surgery of mid-third clavicular fractures is indicated, fixation with a Knowles pin has more advantages than plate fixation.