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Abstract Background The Global initiative for chronic Obstructive Lung Disease (GOLD) defines COPD as a fixed post-bronchodilator ratio of forced expiratory volume in 1 second and forced vital capacity (FEV1/FVC) below 0.7. Age-dependent cut-off values below the lower fifth percentile (LLN) of this ratio derived from the general population have been proposed as an alternative. We wanted to assess the diagnostic accuracy and prognostic capability of the GOLD and LLN definition when compared to an expert-based diagnosis. Methods In a prospective cohort study, 405 patients aged ≥ 65 years with a general practitioner's diagnosis of COPD were recruited and followed up for 4.5 (median; quartiles 3.9; 5.1) years. Prevalence rates of COPD according to GOLD and three LLN definitions and diagnostic performance measurements were calculated. The reference standard was the diagnosis of COPD of an expert panel that used all available diagnostic information, including spirometry and bodyplethysmography. Results Compared to the expert panel diagnosis, 'GOLD-COPD' misclassified 69 (28%) patients, and the three LLNs misclassified 114 (46%), 96 (39%), and 98 (40%) patients, respectively. The GOLD classification led to more false positives, the LLNs to more false negative diagnoses. The main predictors beyond the FEV1/FVC ratio for an expert diagnosis of COPD were the FEV1 % predicted, and the residual volume/total lung capacity ratio (RV/TLC). Adding FEV1 and RV/TLC to GOLD or LLN improved the diagnostic accuracy, resulting in a significant reduction of up to 50% of the number of misdiagnoses. The expert diagnosis of COPD better predicts exacerbations, hospitalizations and mortality than GOLD or LLN. Conclusions GOLD criteria over-diagnose COPD, while LLN definitions under-diagnose COPD in elderly patients as compared to an expert panel diagnosis. Incorporating FEV1 and RV/TLC into the GOLD-COPD or LLN-based definition brings both definitions closer to expert panel diagnosis of COPD, and to daily clinical practice.

Abstract Background Microarray technology allows researchers to simultaneously monitor changes in the expression ratios (ERs) of hundreds of genes and has thereby revolutionized most of biology. Although this technique has the potential of elucidating early stages in an organism's phenotypic response to complex ecological interactions, to date, it has not been fully incorporated into ecological research. This is partially due to a lack of simple procedures of handling and analyzing the expression ratio (ER) data produced from microarrays. Results We describe an analysis of the sources of variation in ERs from 73 hybridized cDNA microarrays, each with 234 herbivory-elicited genes from the model ecological expression system, Nicotiana attenuata, using procedures that are commonly used in ecologic research. Each gene is represented by two independently labeled PCR products and each product was arrayed in quadruplicate. We present a robust method of normalizing and analyzing ERs based on arbitrary thresholds and statistical criteria, and characterize a "norm of reaction" of ERs for 6 genes (4 of known function, 2 of unknown) with different ERs as determined across all analyzed arrays to provide a biologically-informed alternative to the use of arbitrary expression ratios in determining significance of expression. These gene-specific ERs and their variance (gene CV) were used to calculate array-based variances (array CV), which, in turn, were used to study the effects of array age, probe cDNA quantity and quality, and quality of spotted PCR products as estimates of technical variation. Cluster analysis and a Principal Component Analysis (PCA) were used to reveal associations among the transcriptional "imprints" of arrays hybridized with cDNA probes derived from mRNA from N. attenuata plants variously elicited and attacked by different herbivore species and from three congeners: N. quadrivalis, N. longiflora and N. clevelandii. Additionally, the PCA revealed the contribution of individual gene ERs to the associations among arrays. Conclusions While the costs of 'boutique' array fabrication are rapidly declining, familiar methods for the analysis of the data they create are still missing. The case history illustrated here demonstrates the ease with which this powerful technology can be adapted to ecological research.

Arabinogalactan-proteins (AGPs) are a family of hyperglycosylated hydroxyproline-rich cell wall proteins found throughout the plant kingdom. To date, eight Hydroxyproline-galactosyltransferases (Hyp-GALTs), named GALT2-GALT9, are known to catalyze the addition of the first galactose sugar to Hyp residues in AGP protein cores. The generation and characterization of galt23456789 octuple mutants using CRISPR-Cas9 gene editing technology, provided strong reverse genetic evidence that AG glycans are essential for normal vegetative and reproductive growth, as these mutants demonstrated stunted growth, greatly delayed flowering and significant defects in floral organ development and morphogenesis. Compared to the lower seed set of galt25789 quintuple mutants being more so contributed by female gametophytic defects, dramatically low seed-set of octuple mutants was largely due to impaired male reproductive function, specifically due to shorter filaments, delayed anther dehiscence, and large decreases in pollen quantity and viability. Octuple mutant pollen had severely distorted reticulate exine, tectum patterning and intine thickness. Reduced amounts of galactose and arabinose in overall lower amounts of β-Yariv precipitated AGPs illustrated how biological functions of AGPs are affected by abnormal glycosylation., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Author(s).)

It is critical that breweries of all sizes routinely monitor the microbiome of their process to limit financial losses due to microbial contamination. Contamination by beer-spoiling microbes (BSMs) at any point during the brewing process may lead to significant losses for breweries if gone undetected and allowed to spread. Testing and detection of BSMs must be routine and rapid, and because even small breweries need the capability of BSM detection and identification, the method also needs to be affordable. Lactic acid bacteria (LAB) are responsible for most spoilage incidents, many of which have been shown to enter the viable but nonculturable (VBNC) state under conditions present in beer such as cold or oxidative stress. These bacteria are invisible to traditional methods of detection using selective media. This article describes several methods of BSM detection and identification that may be useful in the majority of craft breweries. While there are several genomic methods that meet some or many qualifications of being useful in craft breweries, real-time quantitative polymerase chain reaction (qPCR) currently best meets the desired method characteristics and holds the most utility in this industry, specifically SYBR Green qPCR. qPCR is a targeted method of detection and identification of microbes that is affordable, rapid, specific, sensitive, quantitative, and reliable, and when paired with valid DNA extraction techniques can be used to detect BSMs, including those in the VBNC state., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher., (Copyright © 2023 Oldham and Held.)

Arabinogalactan-proteins (AGPs) are hydroxyproline-rich glycoproteins containing a high sugar content and are widely distributed in the plant kingdom. AGPs have long been suggested to play important roles in sexual plant reproduction. The synthesis of their complex carbohydrates is initiated by a family of hydroxyproline galactosyltransferase (Hyp-GALT) enzymes which add the first galactose to Hyp residues in the protein backbone. Eight Hyp-GALT enzymes have been identified so far, and in the present work a mutant affecting five of these enzymes (galt2galt5galt7galt8galt9) was analyzed regarding the reproductive process. The galt25789 mutant presented a low seed set, and reciprocal crosses indicated a significant female gametophytic contribution to this mutant phenotype. Mutant ovules revealed abnormal callose accumulation inside the embryo sac and integument defects at the micropylar region culminating in defects in pollen tube reception. In addition, immunolocalization and biochemical analyses allowed the detection of a reduction in the amount of glucuronic acid in mutant ovary AGPs. Dramatically low amounts of high-molecular-weight Hyp-O-glycosides obtained following size exclusion chromatography of base-hydrolyzed mutant AGPs compared to the wild type indicated the presence of underglycosylated AGPs in the galt25789 mutant, while the monosaccharide composition of these Hyp-O-glycosides displayed no significant changes compared to the wild-type Hyp-O-glycosides. The present work demonstrates the functional importance of the carbohydrate moieties of AGPs in ovule development and pollen-pistil interactions., (© 2023 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Global gene co-expression networks (GCNs) are powerful tools for functional genomics whereby putative functions and regulatory mechanisms can be inferred by gene co-expression. Cereal crops, such as Hordeum vulgare (barley) and Sorghum bicolor (sorghum), are among the most important plants to civilization. However, co-expression network tools for these plants are lacking. Here, we have constructed global GCNs for barley and sorghum using existing RNA-seq data sets. Meta-information was manually curated and categorized by tissue type to also build tissue-specific GCNs. To enable GCN searching and visualization, we implemented a website and database named PlantNexus. PlantNexus is freely available at https://plantnexus.ohio.edu/., (© The Author(s) 2022. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Arabinogalactan proteins (AGPs) are complex, hyperglycosylated plant cell wall proteins with little known about the biological roles of their glycan moieties in sexual reproduction. Here, we report that GLCAT14A, GLCAT14B, and GLCAT14C, three enzymes responsible for the addition of glucuronic acid residues to AGPs, function in pollen development, polytubey block, and normal embryo development in Arabidopsis. Using biochemical and immunolabeling techniques, we demonstrated that the loss of function of the GLCAT14A, GLCAT14B, and GLCAT14C genes resulted in disorganization of the reticulate structure of the exine wall, abnormal development of the intine layer, and collapse of pollen grains in glcat14a/b and glcat14a/b/c mutants. Synchronous development between locules within the same anther was also lost in some glcat14a/b/c stamens. In addition, we observed excessive attraction of pollen tubes targeting glcat14a/b/c ovules, indicating that the polytubey block mechanism was compromised. Monosaccharide composition analysis revealed significant reductions in all sugars in glcat14a/b and glcat14a/b/c mutants except for arabinose and galactose, while immunolabeling showed decreased amounts of AGP sugar epitopes recognized by glcat14a/b and glcat14a/b/c mutants compared with the wild type. This work demonstrates the important roles that AG glucuronidation plays in Arabidopsis sexual reproduction and reproductive development., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

Background: Arabinogalactan-proteins (AGPs) are structurally complex hydroxyproline-rich cell wall glycoproteins ubiquitous in the plant kingdom. AGPs biosynthesis involves a series of post-translational modifications including the addition of type II arabinogalactans to non-contiguous Hyp residues. To date, eight Hyp-galactosyltransferases (Hyp-GALTs; GALT2-GALT9) belonging to CAZy GT31, are known to catalyze the addition of the first galactose residues to AGP protein backbones and enable subsequent AGP glycosylation. The extent of genetic redundancy, however, remains to be elucidated for the Hyp-GALT gene family., Results: To examine their gene redundancy and functions, we generated various multiple gene knock-outs, including a triple mutant (galt5 galt8 galt9), two quadruple mutants (galt2 galt5 galt7 galt8, galt2 galt5 galt7 galt9), and one quintuple mutant (galt2 galt5 galt7 galt8 galt9), and comprehensively examined their biochemical and physiological phenotypes. The key findings include: AGP precipitations with β-Yariv reagent showed that GALT2, GALT5, GALT7, GALT8 and GALT9 act redundantly with respect to AGP glycosylation in cauline and rosette leaves, while the activity of GALT7, GALT8 and GALT9 dominate in the stem, silique and flowers. Monosaccharide composition analysis showed that galactose was decreased in the silique and root AGPs of the Hyp-GALT mutants. TEM analysis of 25789 quintuple mutant stems indicated cell wall defects coincident with the observed developmental and growth impairment in these Hyp-GALT mutants. Correlated with expression patterns, galt2, galt5, galt7, galt8, and galt9 display equal additive effects on insensitivity to β-Yariv-induced growth inhibition, silique length, plant height, and pollen viability. Interestingly, galt7, galt8, and galt9 contributed more to primary root growth and root tip swelling under salt stress, whereas galt2 and galt5 played more important roles in seed morphology, germination defects and seed set. Pollen defects likely contributed to the reduced seed set in these mutants., Conclusion: Additive and pleiotropic effects of GALT2, GALT5, GALT7, GALT8 and GALT9 on vegetative and reproductive growth phenotypes were teased apart via generation of different combinations of Hyp-GALT knock-out mutants. Taken together, the generation of higher order Hyp-GALT mutants demonstrate the functional importance of AG polysaccharides decorating the AGPs with respect to various aspects of plant growth and development., (© 2021. The Author(s).)

Transcriptional regulatory mechanisms governing plant cell wall biosynthesis are incomplete. Expression programs that activate wall biosynthesis are well understood, but mechanisms that control the attenuation of gene expression networks remain elusive. Previous work has shown that small RNAs (sRNAs) derived from the HvCESA 6 ( Hordeum vulgare , Hv ) antisense transcripts are naturally produced and are capable of regulating aspects of wall biosynthesis. Here, we further test the hypothesis that CESA -derived sRNAs generated from CESA antisense transcripts are involved in the regulation of cellulose and broader cell wall biosynthesis. Antisense transcripts were detected for some but not all members of the CESA gene family in both barley and Brachypodium distachyon . Phylogenetic analysis indicates that antisense transcripts are detected for most primary cell wall CESA genes, suggesting a possible role in the transition from primary to secondary cell wall biosynthesis. Focusing on one antisense transcript, HvCESA1 shows dynamic expression throughout development, is correlated with corresponding sRNAs over the same period and is anticorrelated with HvCESA1 mRNA expression. To assess the broader impacts of CESA -derived sRNAs on the regulation of cell wall biosynthesis, transcript profiling was performed on barley tissues overexpressing CESA -derived sRNAs. Together, the data support the hypothesis that CESA antisense transcripts function through an RNA-induced silencing mechanism, to degrade cis transcripts, and may also trigger trans -acting silencing on related genes to alter the expression of cell wall gene networks., Competing Interests: The authors declare no conflict of interest associated with the work described in this manuscript., (© 2021 The Authors. Plant Direct published by American Society of Plant Biologists and the Society for Experimental Biology and John Wiley & Sons Ltd.)

Arabinogalactan proteins (AGPs) contain arabinogalactan (AG) polysaccharides that are biologically relevant to plant growth processes. Here, the biochemical and physiological roles of three Golgi localized β-glucuronosyltransferase genes ( GLCAT14A , GLCAT14B and GLCAT14C ) in Arabidopsis thaliana, responsible for the addition of glucuronic acid to AG chains, were further investigated using single, double and triple glcat14 mutant plants. These proteins were localized to the Golgi apparatus when transiently expressed in Nicotiana benthamiana . Sugar analysis of AGP extracts from Arabidopsis stem, leaf and siliques showed a consistent reduction in glucuronic acid in glcat14 mutants relative to wild type, with concomitant effects resulting in tissue-specific alterations, especially in arabinose and galactose sugars. Although we observed defects in trichome branching in glca14a/b and glca14a/b/c mutants, scanning electron microscope analysis/energy dispersive microanalysis (SEM/EDX) showed no difference in the calcium content of trichomes in these mutants relative to wild type. Immunoblot analyses of the stem and leaf showed a reduction in AGPs as detected with the LM2 antibody in glcat14a/b and glcat14a/b/c mutants relative to wild type. The current work exemplifies the possibility of conducting structure-function assessment of cell wall biosynthetic genes to identify their physiological roles in plant growth and development.

Background: Arabinogalactan-proteins (AGPs) are heavily glycosylated with type II arabinogalactan (AG) polysaccharides attached to hydroxyproline residues in their protein backbone. Type II AGs are necessary for plant growth and critically important for the establishment of normal cellular functions. Despite the importance of type II AGs in plant development, our understanding of the underlying role of these glycans/sugar residues in mucilage formation and seed coat epidermal cell development is poorly understood and far from complete. One such sugar residue is the glucuronic acid residues of AGPs that are transferred onto AGP glycans by the action of β-glucuronosyltransferase genes/enzymes., Results: Here, we have characterized two β-glucuronosyltransferase genes, GLCAT14A and GLCAT14C, that are involved in the transfer of β-glucuronic acid (GlcA) to type II AGs. Using a reverse genetics approach, we observed that glcat14a-1 mutants displayed subtle alterations in mucilage pectin homogalacturonan (HG) compared to wild type (WT), while glcat14a-1glcat14c-1 mutants displayed much more severe mucilage phenotypes, including loss of adherent mucilage and significant alterations in cellulose ray formation and seed coat morphology. Monosaccharide composition analysis showed significant alterations in the sugar amounts of glcat14a-1glcat14c-1 mutants relative to WT in the adherent and non-adherent seed mucilage. Also, a reduction in total mucilage content was observed in glcat14a-1glcat14c-1 mutants relative to WT. In addition, glcat14a-1glcat14c-1 mutants showed defects in pectin formation, calcium content and the degree of pectin methyl-esterification (DM) as well as reductions in crystalline cellulose content and seed size., Conclusions: These results raise important questions regarding cell wall polymer interactions and organization during mucilage formation. We propose that the enzymatic activities of GLCAT14A and GLCAT14C play partially redundant roles and are required for the organization of the mucilage matrix and seed size in Arabidopsis thaliana. This work brings us a step closer towards identifying potential gene targets for engineering plant cell walls for industrial applications.

The extensin (EXT) network is elaborated by the covalent intermolecular crosslinking of EXT glycoprotein monomers, and its proper assembly is important for numerous aspects of basic wall architecture and cellular defense. In this review, we discuss new advances in the secretion of EXT monomers and the molecular drivers of EXT network self-assembly. Many of the functions of EXTs are conferred through covalent crosslinking into the wall, so we also discuss the different types of known intermolecular crosslinks, the enzymes that are involved, as well as the potential for additional crosslinks that are yet to be identified. EXTs also function in wall architecture independent of crosslinking status, and therefore, we explore the role of non-crosslinking EXTs. As EXT crosslinking is upregulated in response to wounding and pathogen infection, we discuss a potential regulatory mechanism to control covalent crosslinking and its relationship to the subcellular localization of the crosslinking enzymes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mishler-Elmore, Zhou, Sukul, Oblak, Tan, Faik and Held.)

Background: Arabinogalactan-proteins (AGPs) are a class of hydroxyproline-rich proteins (HRGPs) that are heavily glycosylated (> 90%) with type II arabinogalactans (AGs). AGPs are implicated in various plant growth and development processes including cell expansion, somatic embryogenesis, root and stem growth, salt tolerance, hormone signaling, male and female gametophyte development, and defense. To date, eight Hyp-O-galactosyltransferases (GALT2-6, HPGT1-3) have been identified; these enzymes are responsible for adding the first sugar, galactose, onto AGPs. Due to gene redundancy among the GALTs, single or double galt genetic knockout mutants are often not sufficient to fully reveal their biological functions., Results: Here, we report the successful application of CRISPR-Cas9 gene editing/multiplexing technology to generate higher-order knockout mutants of five members of the GALT gene family (GALT2-6). AGPs analysis of higher-order galt mutants (galt2 galt5, galt3 galt4 galt6, and galt2 galt3 galt4 galt5 gal6) demonstrated significantly less glycosylated AGPs in rosette leaves, stems, and siliques compared to the corresponding wild-type organs. Monosaccharide composition analysis of AGPs isolated from rosette leaves revealed significant decreases in arabinose and galactose in all the higher-order galt mutants. Phenotypic analyses revealed that mutation of two or more GALT genes was able to overcome the growth inhibitory effect of β-D-Gal-Yariv reagent, which specifically binds to β-1,3-galactan backbones on AGPs. In addition, the galt2 galt3 galt4 galt5 gal6 mutant exhibited reduced overall growth, impaired root growth, abnormal pollen, shorter siliques, and reduced seed set. Reciprocal crossing experiments demonstrated that galt2 galt3 galt4 galt5 gal6 mutants had defects in the female gametophyte which were responsible for reduced seed set., Conclusions: Our CRISPR/Cas9 gene editing/multiplexing approach provides a simpler and faster way to generate higher-order mutants for functional characterization compared to conventional genetic crossing of T-DNA mutant lines. Higher-order galt mutants produced and characterized in this study provide insight into the relationship between sugar decorations and the various biological functions attributed to AGPs in plants.

Agave americana L. is a highly productive, drought-tolerant species being investigated as a feedstock for biofuel production. Some Agave spp. yield crop biomass in semi-arid conditions that are comparable to C 3 and C 4 crops grown in areas with high rainfall. This study evaluates the bioethanol yield potential of A. americana by (1) examining the relationship between water use efficiency (WUE) and plant carbohydrates, (2) quantifying the carbohydrate and energy content of the plant tissue, and (3) comparing the products of enzymatic hydrolysis to that of other candidate feedstocks ( Miscanthus x giganteus Greef et Deuter, Sorghum bicolor (L.) Moench, and Panicum virgatum L.). Results indicate that (1) WUE does not significantly affect soluble and insoluble (i.e., structural) carbohydrate composition per unit mass in A. americana ; (2) without pretreatment, A. americana biomass had the lowest gross heat of combustion, or higher heating/calorific value, compared to high yielding C 4 crops; and (3) after separation of soluble carbohydrates, A. americana cellulosic biomass was most easily hydrolyzed by enzymes with greater sugar yield per unit mass compared to the other biomass feedstocks. These results indicate that A. americana can produce substantial yields of soluble carbohydrates with minimal water inputs required for cultivation, and fiber portions of the crop can be readily deconstructed by cellulolytic enzymes for subsequent biochemical fermentation., (Copyright © 2020 Jones, Zhou, Held and Davis.)

Background: Arabinogalactan-proteins (AGPs) are one of the most complex protein families in the plant kingdom and are present in the cell walls of all land plants. AGPs are implicated in diverse biological processes such as plant growth, development, reproduction, and stress responses. AGPs are extensively glycosylated by the addition of type II arabinogalactan (AG) polysaccharides to hydroxyproline residues in their protein cores. Glucuronic acid (GlcA) is the only negatively charged sugar added to AGPs and the functions of GlcA residues on AGPs remain to be elucidated., Results: Three members of the CAZy GT14 family (GLCAT14A-At5g39990, GLCAT14B-At5g15050, and GLCAT14C-At2g37585), which are responsible for transferring glucuronic acid (GlcA) to AGPs, were functionally characterized using a CRISPR/Cas9 gene editing approach in Arabidopsis. RNA seq and qRT-PCR data showed all three GLCAT genes were broadly expressed in different plant tissues, with GLCAT14A and GLCAT14B showing particularly high expression in the micropylar endosperm. Biochemical analysis of the AGPs from knock-out mutants of various glcat single, double, and triple mutants revealed that double and triple mutants generally had small increases of Ara and Gal and concomitant reductions of GlcA, particularly in the glcat14a glcat14b and glcat14a glcat14b glcat14c mutants. Moreover, AGPs isolated from all the glcat mutants displayed significant reductions in calcium binding compared to WT. Further phenotypic analyses found that the glcat14a glcat14b and glcat14a glcat14b glcat14c mutants exhibited significant delays in seed germination, reductions in root hair length, reductions in trichome branching, and accumulation of defective pollen grains. Additionally, both glcat14b glcat14c and glcat14a glcat14b glcat14c displayed significantly shorter siliques and reduced seed set. Finally, all higher-order mutants exhibited significant reductions in adherent seed coat mucilage., Conclusions: This research provides genetic evidence that GLCAT14A-C function in the transfer of GlcA to AGPs, which in turn play a role in a variety of biochemical and physiological phenotypes including calcium binding by AGPs, seed germination, root hair growth, trichome branching, pollen development, silique development, seed set, and adherent seed coat mucilage accumulation.

The formation of erythroid progenitor cells depends sharply upon erythropoietin (EPO), its cell surface receptor (erythropoietin receptor, EPOR), and Janus kinase 2 (JAK2). Clinically, recombinant human EPO (rhEPO) additionally is an important anti-anemia agent for chronic kidney disease (CKD), myelodysplastic syndrome (MDS) and chemotherapy, but induces hypertension, and can exert certain pro-tumorigenic effects. Cellular signals transduced by EPOR/JAK2 complexes, and the nature of EPO-modulated signal transduction factors, therefore are of significant interest. By employing phospho-tyrosine post-translational modification (p-Y PTM) proteomics and human EPO- dependent UT7epo cells, we have identified 22 novel kinases and phosphatases as novel EPO targets, together with their specific sites of p-Y modification. New kinases modified due to EPO include membrane palmitoylated protein 1 (MPP1) and guanylate kinase 1 (GUK1) guanylate kinases, together with the cytoskeleton remodeling kinases, pseudopodium enriched atypical kinase 1 (PEAK1) and AP2 associated kinase 1 (AAK1). Novel EPO- modified phosphatases include protein tyrosine phosphatase receptor type A (PTPRA), phosphohistidine phosphatase 1 (PHPT1), tensin 2 (TENC1), ubiquitin associated and SH3 domain containing B (UBASH3B) and protein tyrosine phosphatase non-receptor type 18 (PTPN18). Based on PTPN18's high expression in hematopoietic progenitors, its novel connection to JAK kinase signaling, and a unique EPO- regulated PTPN18-pY389 motif which is modulated by JAK2 inhibitors, PTPN18's actions in UT7epo cells were investigated. Upon ectopic expression, wt-PTPN18 promoted EPO dose-dependent cell proliferation, and survival. Mechanistically, PTPN18 sustained the EPO- induced activation of not only mitogen-activated protein kinases 1 and 3 (ERK1/2), AKT serine/threonine kinase 1-3 (AKT), and signal transducer and activator of transcription 5A and 5B (STAT5), but also JAK2. Each effect further proved to depend upon PTPN18's EPO- modulated (p)Y389 site. In analyses of the EPOR and the associated adaptor protein RHEX (regulator of hemoglobinization and erythroid cell expansion), wt-PTPN18 increased high molecular weight EPOR forms, while sharply inhibiting the EPO-induced phosphorylation of RHEX-pY141. Each effect likewise depended upon PTPN18-Y389. PTPN18 thus promotes signals for EPO-dependent hematopoietic cell growth, and may represent a new druggable target for myeloproliferative neoplasms., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Erythroid cell formation critically depends on signals transduced via erythropoietin (EPO)/EPO receptor (EPOR)/JAK2 complexes. This includes not only core response modules (e.g., JAK2/STAT5, RAS/MEK/ERK), but also specialized effectors (e.g., erythroferrone, ASCT2 glutamine transport, Spi2A). By using phospho-proteomics and a human erythroblastic cell model, we identify 121 new EPO target proteins, together with their EPO-modulated domains and phosphosites. Gene ontology (GO) enrichment for "Molecular Function" identified adaptor proteins as one top EPO target category. This includes a novel EPOR/JAK2-coupled network of actin assemblage modifiers, with adaptors DLG-1, DLG-3, WAS, WASL, and CD2AP as prime components. "Cellular Component" GO analysis further identified 19 new EPO-modulated cytoskeletal targets including the erythroid cytoskeletal targets spectrin A, spectrin B, adducin 2, and glycophorin C. In each, EPO-induced phosphorylation occurred at pY sites and subdomains, which suggests coordinated regulation by EPO of the erythroid cytoskeleton. GO analysis of "Biological Processes" further revealed metabolic regulators as a likewise unexpected EPO target set. Targets included aldolase A, pyruvate dehydrogenase α1, and thioredoxin-interacting protein (TXNIP), with EPO-modulated p-Y sites in each occurring within functional subdomains. In TXNIP, EPO-induced phosphorylation occurred at novel p-T349 and p-S358 sites, and was paralleled by rapid increases in TXNIP levels. In UT7epo-E and primary human stem cell (HSC)-derived erythroid progenitor cells, lentivirus-mediated short hairpin RNA knockdown studies revealed novel pro-erythropoietic roles for TXNIP. Specifically, TXNIP's knockdown sharply inhibited c-KIT expression; compromised EPO dose-dependent erythroblast proliferation and survival; and delayed late-stage erythroblast formation. Overall, new insight is provided into EPO's diverse action mechanisms and TXNIP's contributions to EPO-dependent human erythropoiesis., Competing Interests: Conflict of interest disclosure The authors declare no competing interests., (Copyright © 2020 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Mesenchymal stem-like (MSL) breast cancers are enriched for cells with tumor reconstituting and mesenchymal characteristics. These cancers are often triple-negative and have a poor prognosis. Few effective targeted treatment options exist for patients with these cancers, and even when targeted therapies exist, resistance often arises and tumors recur, due in part to drug-tolerant persisting tumor cells with self-renewal capability. Effective treatment strategies will combine agents that target the bulk-tumor and reconstituting cells. In order to identify such a combination therapy, we conducted an inhibitor screen using 40 targeted agents at three different doses in all pairwise combinations. Checkpoint Kinase 1 (CHK1) inhibitors were identified as potent inhibitors of MSL breast cancers. When combined with a pro-apoptotic agent/B Cell Lymphoma 2 (BCL2) inhibitor, the effectiveness of the combination regimen was super-additive compared to either treatment alone and was selective for MSL cancers. Treatment of MSL breast cancer cells results in DNA damage, cell-cycle defects characterized by a prolonged S-phase, increased apoptosis and decreased colony forming abilities compared to untreated cells. These data suggest that a combination of a CHK1 and BCL2 inhibitor could be an effective treatment for patients with MSL breast cancer. Several other effective drug combinations were also identified., Competing Interests: CONFLICTS OF INTEREST The authors declare that they have no competing interests.

Occam's Razor suggests a new model of pollen tube tip growth based on a novel Hechtian oscillator that integrates a periplasmic arabinogalactan glycoprotein-calcium (AGP-Ca 2+ ) capacitor with tip-localized AGPs as the source of tip-focussed cytosolic Ca 2+ oscillations: Hechtian adhesion between the plasma membrane and the cell wall of the growing tip acts as a piconewton force transducer that couples the internal stress of a rapidly growing wall to the plasma membrane. Such Hechtian transduction opens stretch-activated Ca 2+ channels and activates H + -ATPase proton pump efflux that dissociates periplasmic AGP-Ca 2+ resulting in a Ca 2+ influx that activates exocytosis of wall precursors. Thus, a highly simplified pectic primary cell wall regulates its own synthesis by a Hechtian growth oscillator that regulates overall tip growth. By analogy with the three cryptic inscriptions of the classical Rosetta Stone, the Hechtian Hypothesis translates classical AGP function as a Ca 2+ capacitor, pollen tube guide and wall plasticizer into a simple but widely applicable model of tip growth. Even wider ramifications of the Hechtian oscillator may implicate AGPs in osmosensing or gravisensing and other tropisms, leading us yet further towards the Holy Grail of plant growth., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

Personalized cancer therapy is based on a patient's tumor lineage, histopathology, expression analyses, and/or tumor DNA or RNA analysis. Here, we aim to develop an in vitro functional assay of a patient's living cancer cells that could complement these approaches. We present methods for developing cell cultures from tumor biopsies and identify the types of samples and culture conditions associated with higher efficiency of model establishment. Toward the application of patient-derived cell cultures for personalized care, we established an immunofluorescence-based functional assay that quantifies cancer cell responses to targeted therapy in mixed cell cultures. Assaying patient-derived lung cancer cultures with this method showed promise in modeling patient response for diagnostic use. This platform should allow for the development of co-clinical trial studies to prospectively test the value of drug profiling on tumor-biopsy-derived cultures to direct patient care., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Pancreatic adenocarcinoma (PDAC) is the fourth most common cause of cancer-related death in the United States. PDAC is difficult to manage effectively, with a five-year survival rate of only 5%. PDAC is largely driven by activating KRAS mutations, and as such, cannot be directly targeted with therapeutic agents that affect the activated protein. Instead, inhibition of downstream signaling and other targets will be necessary to effectively manage PDAC. Here, we describe a tiered single-agent and combination compound screen to identify targeted agents that impair growth of a panel of PDAC cell lines. Several of the combinations identified from the screen were further validated for efficacy and mechanism. Combination of the bromodomain inhibitor JQ1 and the neddylation inhibitor MLN4294 altered the production of reactive oxygen species in PDAC cells, ultimately leading to defects in the DNA damage response. Dual bromodomain/neddylation blockade inhibited in vivo growth of PDAC cell line xenografts. Overall, this work revealed novel combinatorial regimens, including JQ1 plus MLN4294, which show promise for the treatment of RAS -driven PDAC. Mol Cancer Ther; 16(6); 1041-53. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Triple-negative breast cancer (TNBC) remains an aggressive disease without effective targeted therapies. In this study, we addressed this challenge by testing 128 FDA-approved or investigational drugs as either single agents or in 768 pairwise drug combinations in TNBC cell lines to identify synergistic combinations tractable to clinical translation. Medium-throughput results were scrutinized and extensively analyzed for sensitivity patterns, synergy, anticancer activity, and were validated in low-throughput experiments. Principal component analysis revealed that a fraction of all upregulated or downregulated genes of a particular targeted pathway could partly explain cell sensitivity toward agents targeting that pathway. Combination therapies deemed immediately tractable to translation included ABT-263/crizotinib, ABT-263/paclitaxel, paclitaxel/JQ1, ABT-263/XL-184, and paclitaxel/nutlin-3, all of which exhibited synergistic antiproliferative and apoptotic activity in multiple TNBC backgrounds. Mechanistic investigations of the ABT-263/crizotinib combination offering a potentially rapid path to clinic demonstrated RTK blockade, inhibition of mitogenic signaling, and proapoptotic signal induction in basal and mesenchymal stem-like TNBC. Our findings provide preclinical proof of concept for several combination treatments of TNBC, which offer near-term prospects for clinical translation. Cancer Res; 77(2); 566-78. ©2016 AACR., (©2016 American Association for Cancer Research.)

The complexity of cell wall composition and structure determines the strength, flexibility, and function of the primary cell wall in plants. However, the contribution of the various components to cell wall integrity (CWI) and function remains unclear. Modifications of cell wall composition can induce plant responses known as CWI control. In this study, we used transgenic expression of the fungal feruloyl esterase AnFAE to examine the effect of post-synthetic modification of Arabidopsis and Brachypodium cell walls. Transgenic Arabidopsis plants expressing AnFAE showed a significant reduction of monomeric ferulic acid, decreased amounts of wall-associated extensins, and increased susceptibility to Botrytis cinerea, compared with wild type. Transgenic Brachypodium showed reductions in monomeric and dimeric ferulic acids and increased susceptibility to Bipolaris sorokiniana. Upon infection, transgenic Arabidopsis and Brachypodium plants also showed increased expression of several defense-related genes compared with wild type. These results demonstrate a role, in both monocot and dicot plants, of polysaccharide feruloylation in plant CWI, which contributes to plant resistance to necrotrophic pathogens.

A new coupling of electrochemistry with mass spectrometry (MS) using probe electrospray ionization (PESI) is presented. Due to the high salt tolerance of PESI, the detection of electrochemical reaction products in room-temperature ionic liquids (RTILs) is realized for the first time. Furthermore, PESI-MS allows the analysis of electrochemical reaction products on different or multiple electrode surfaces. In addition, peptides and proteins fractionated through isoelectric focusing (IEF) in the presence of an external electric field can also be directly analyzed by using PESI-MS, suggesting a new and rapid characterization means for the IEF technique. This study reveals the versatility of EC/PESI-MS, which could have an impact in electrochemistry and bioanalysis fields., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Targeting anti-apoptotic proteins can sensitize tumor cells to conventional chemotherapies or other targeted agents. Antagonizing the Inhibitor of Apoptosis Proteins (IAPs) with mimetics of the pro-apoptotic protein SMAC is one such approach. We used sensitization compound screening to uncover possible agents with the potential to further sensitize lung adenocarcinoma cells to the SMAC mimetic Debio 1143. Several compounds in combination with Debio 1143, including taxanes, topoisomerase inhibitors, and bromodomain inhibitors, super-additively inhibited growth and clonogenicity of lung adenocarcinoma cells. Co-treatment with Debio 1143 and the bromodomain inhibitor JQ1 suppresses the expression of c-IAP1, c-IAP2, and XIAP. Non-canonical NF-κB signaling is also activated following Debio 1143 treatment, and Debio 1143 induces the formation of the ripoptosome in Debio 1143-sensitive cell lines. Sensitivity to Debio 1143 and JQ1 co-treatment was associated with baseline caspase-8 expression. In vivo treatment of lung adenocarcinoma xenografts with Debio 1143 in combination with JQ1 or docetaxel reduced tumor volume more than either single agent alone. As Debio 1143-containing combinations effectively inhibited both in vitro and in vivo growth of lung adenocarcinoma cells, these data provide a rationale for Debio 1143 combinations currently being evaluated in ongoing clinical trials and suggest potential utility of other combinations identified here.

Extensins (EXTs) are hydroxyproline-rich glycoproteins (HRGPs) that are structural components of the plant primary cell wall. They are basic proteins and are highly glycosylated with carbohydrate accounting for >50% of their dry weight. Carbohydrate occurs as monogalactosyl serine and arabinosyl hydroxyproline, with arabinosides ranging in size from ~1 to 4 or 5 residues. Proposed functions of EXT arabinosylation include stabilizing the polyproline II helix structure and facilitating EXT cross-linking. Here, the involvement of arabinosylation in EXT cross-linking was investigated by assaying the initial cross-linking rate and degree of cross-linking of partially or fully de-arabinosylated EXTs using an in vitro cross-linking assay followed by gel permeation chromatography. Our results indicate that EXT arabinosylation is required for EXT cross-linking in vitro and the fourth arabinosyl residue in the tetraarabinoside chain, which is uniquely α-linked, may determine the initial cross-linking rate. Our results also confirm the conserved structure of the oligoarabinosides across species, indicating an evolutionary significance for EXT arabinosylation.

BRAF inhibitors have revolutionized treatment of mutant BRAF metastatic melanomas. However, resistance develops rapidly following BRAF inhibitor treatment. We have found that BRAF-mutant melanoma cell lines are more sensitive than wild-type BRAF cells to the small molecule tyrosine kinase inhibitor dovitinib. Sensitivity is associated with inhibition of a series of known dovitinib targets. Dovitinib in combination with several agents inhibits growth more effectively than either agent alone. These combinations inhibit BRAF-mutant melanoma and colorectal carcinoma cell lines, including cell lines with intrinsic or selected BRAF inhibitor resistance. Hence, combinations of dovitinib with second agents are potentially effective therapies for BRAF-mutant melanomas, regardless of their sensitivity to BRAF inhibitors., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

BRAF kinase inhibitors have dramatically affected treatment of BRAF(V600E) (/) (K)-driven metastatic melanoma. Early responses assessed using [(18)F]fluorodeoxyglucose uptake-positron emission tomography (FDG-PET) have shown dramatic reduction of radiotracer signal within 2 weeks of treatment. Despite high response rates, relapse occurs in nearly all cases, frequently at sites of treated metastatic disease. It remains unclear whether initial loss of (18)FDG uptake is due to tumor cell death or other reasons. Here, we provide evidence of melanoma cell volume reduction in a patient cohort treated with BRAF inhibitors. We present data demonstrating that BRAF inhibition reduces melanoma glucose uptake per cell, but that this change is no longer significant following normalization for cell volume changes. We also demonstrate that volume normalization greatly reduces differences in transmembrane glucose transport and hexokinase-mediated phosphorylation. Mechanistic studies suggest that this loss of cell volume is due in large part to decreases in new protein translation as a consequence of vemurafenib treatment. Ultimately, our findings suggest that cell volume regulation constitutes an important physiologic parameter that may significantly contribute to radiographic changes observed in clinic., (©2015 American Association for Cancer Research.)

Pectins are critical polysaccharides of the cell wall that are involved in key aspects of a plant's life, including cell-wall stiffness, cell-to-cell adhesion, and mechanical strength. Pectins undergo methylesterification, which affects their cellular roles. Pectin methyltransferases are believed to methylesterify pectins in the Golgi, but little is known about their identity. To date, there is only circumstantial evidence to support a role for QUASIMODO2 (QUA2)-like proteins and an unrelated plant-specific protein, cotton Golgi-related 3 (CGR3), in pectin methylesterification. To add to the knowledge of pectin biosynthesis, here we characterized a close homolog of CGR3, named CGR2, and evaluated the effect of loss-of-function mutants and over-expression lines of CGR2 and CGR3 in planta. Our results show that, similar to CGR3, CGR2 is a Golgi protein whose enzyme active site is located in the Golgi lumen where pectin methylesterification occurs. Through phenotypical analyses, we also established that simultaneous loss of CGR2 and CGR3 causes severe defects in plant growth and development, supporting critical but overlapping functional roles of these proteins. Qualitative and quantitative cell-wall analytical assays of the double knockout mutant demonstrated reduced levels of pectin methylesterification, coupled with decreased microsomal pectin methyltransferase activity. Conversely, CGR2 and CGR3 over-expression lines have markedly opposite phenotypes to the double knockout mutant, with increased cell-wall methylesterification levels and microsomal pectin methyltransferase activity. Based on these findings, we propose that CGR2 and CGR3 are critical proteins in plant growth and development that act redundantly in pectin methylesterification in the Golgi apparatus., (© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.)

The regulation of plant cell growth and early defense response involves the insolubilization of hydroxyproline-rich glycoproteins (HRGPs), such as extensin, in the primary cell wall. In tomato (Lycopersicon esculentum), insolubilization occurs by the formation of tyrosyl-crosslinks catalyzed specifically by the pI 4.6 extensin peroxidase (EP). To date, neither the gene encoding EP nor the protein itself has been identified. Here, we have identified tomato EP candidates using both proteomic and bioinformatic approaches. Bioinformatic screening of the tomato genome yielded eight EP candidates, which contained a putative signal sequence and a predicted pI near 4.6. Biochemical fractionation of tomato culture media followed by proteomic detection further refined our list of EP candidates to three, with the lead candidate designated (CG5). To test for EP crosslinking activity, we cloned into a bacterial expression vector the CG5 open-reading frame from tomato cDNA. The CG5 was expressed in Escherichia coli, fractionated from inclusion bodies, and folded in vitro. The peroxidase activity of CG5 was assayed and quantified by ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) assay. Subsequent extensin crosslinking assays showed that CG5 can covalently crosslink authentic tomato P1 extensin and P3-type extensin analogs in vitro supporting our hypothesis that CG5 encodes a tomato EP., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Extensins are members of the cell wall hydroxyproline-rich glycoprotein (HRGP) superfamily that form covalently cross-linked networks in primary cell walls. A knockout mutation in EXT3 (AT1G21310), the gene coding EXTENSIN 3 (EXT3) in Arabidopsis Landsberg erecta resulted in a lethal phenotype, although about 20% of the knockout plants have an apparently normal phenotype (ANP). In this study the root cell wall HRGP components of wild-type, ANP and the ext3 mutant seedlings were characterized by peptide fractionation of trypsin digested anhydrous hydrogen fluoride deglycosylated wall residues and by sequencing using LC-MS/MS. Several HRGPs, including EXT3, were identified in the wild-type root walls but not in walls of the ANP and lethal mutant. Indeed the ANP walls and walls of mutants displaying the lethal phenotype possessed HRGPs, but the profiles suggest that changes in the amount and perhaps type may account for the corresponding phenotypes.

Biotechnological approaches to reduce or modify lignin in biomass crops are predicated on the assumption that it is the principal determinant of the recalcitrance of biomass to enzymatic digestion for biofuels production. We defined quantitative trait loci (QTL) in the Intermated B73 × Mo17 recombinant inbred maize (Zea mays) population using pyrolysis molecular-beam mass spectrometry to establish stem lignin content and an enzymatic hydrolysis assay to measure glucose and xylose yield. Among five multiyear QTL for lignin abundance, two for 4-vinylphenol abundance, and four for glucose and/or xylose yield, not a single QTL for aromatic abundance and sugar yield was shared. A genome-wide association study for lignin abundance and sugar yield of the 282-member maize association panel provided candidate genes in the 11 QTL of the B73 and Mo17 parents but showed that many other alleles impacting these traits exist among this broader pool of maize genetic diversity. B73 and Mo17 genotypes exhibited large differences in gene expression in developing stem tissues independent of allelic variation. Combining these complementary genetic approaches provides a narrowed list of candidate genes. A cluster of SCARECROW-LIKE9 and SCARECROW-LIKE14 transcription factor genes provides exceptionally strong candidate genes emerging from the genome-wide association study. In addition to these and genes associated with cell wall metabolism, candidates include several other transcription factors associated with vascularization and fiber formation and components of cellular signaling pathways. These results provide new insights and strategies beyond the modification of lignin to enhance yields of biofuels from genetically modified biomass., (© 2014 American Society of Plant Biologists. All Rights Reserved.)

Owing to high petroleum prices, there has been a major push in recent years to use lignocellulosic biomass as biorefinery feedstocks. Unfortunately, by nature's design, lignocellulosic biomass is notoriously recalcitrant. Cellulose is the most abundant renewable carbon source on the planet and comprises glucan polysaccharides which self-assemble into paracrystalline microfibrils. The extent of cellulose crystallinity largely contributes to biomass recalcitrance. Additionally, cellulose microfibrils are embedded into both hemicellulose and lignin polymeric networks, making cellulose accessibility an additional obstacle. Pretreatment is necessary before enzymatic hydrolysis in order to liberate high yields of glucose and other fermentable sugars from biomass polysaccharides. This work discusses two pretreatment methods, supercritical CO2 and ionic liquids (ILs). Both methods utilize green solvents that do not emit toxic vapours. Mechanisms for destroying or weakening biomass recalcitrance have been explored. Various pretreatment operating parameters such as temperature, pressure, time, dry biomass/solvent ratio, water content, etc. have been investigated for the pretreatment of various biomass types such as corn stover, switchgrass, sugarcane bagasse, soft and hard wood. The two pretreatment methods have their pros and cons. For example, supercritical CO2 explosion pretreatment uses inexpensive CO2, but requires a high pressure. By comparison, while IL pretreatment does not require an elevated pressure, ILs are still too expensive for large-scale uses. Further research and development are needed to make the two green pretreatment methods practical.

Unlabelled: Resistance and partial responses to targeted monotherapy are major obstacles in cancer treatment. Systematic approaches to identify efficacious drug combinations for cancer are not well established, especially in the context of genotype. To address this, we have tested pairwise combinations of an array of small-molecule inhibitors on early-passage melanoma cultures using combinatorial drug screening. Results reveal several inhibitor combinations effective for melanomas with activating RAS or BRAF mutations, including mutant BRAF melanomas with intrinsic or acquired resistance to vemurafenib. Inhibition of both EGF receptor and AKT sensitized treatment-resistant BRAF mutant melanoma cultures to vemurafenib. Melanomas with RAS mutations were more resistant to combination therapies relative to BRAF mutants, but were sensitive to combinations of statins and cyclin-dependent kinase inhibitors in vitro and in vivo. These results show the use of combinatorial drug screening for discovering unique treatment regimens that overcome resistance phenotypes of mutant BRAF- and RAS-driven melanomas., Significance: We have used drug combinatorial screening to identify effective combinations for mutant BRAF melanomas, including those resistant to vemurafenib, and mutant RAS melanomas that are resistant to many therapies. Mechanisms governing the interactions of the drug combinations are proposed, and in vivo xenografts show the enhanced benefit and tolerability of a mutant RAS -selective combination, which is currently lacking in the clinic.

Eukaryotic cells use COPII-coated carriers for endoplasmic reticulum (ER)-to-Golgi protein transport. Selective cargo capture into ER-derived carriers is largely driven by the SEC24 component of the COPII coat. The Arabidopsis genome encodes three AtSEC24 genes with overlapping expression profiles but it is yet to be established whether the AtSEC24 proteins have overlapping roles in plant growth and development. Taking advantage of Arabidopsis thaliana as a model plant system for studying gene function in vivo, through reciprocal crosses, pollen characterization, and complementation tests, evidence is provided for a role for AtSEC24A in the male gametophyte. It is established that an AtSEC24A loss-of-function mutation is tolerated in the female gametophyte but that it causes defects in pollen leading to failure of male transmission of the AtSEC24A mutation. These data provide a characterization of plant SEC24 family in planta showing incompletely overlapping functions of the AtSEC24 isoforms. The results also attribute a novel role to SEC24 proteins in a multicellular model system, specifically in male fertility.

Plant cell walls are complex structures that offer structural and mechanical support to plant cells and are ultimately responsible for plant architecture and form. Pectins are a large group of complex polysaccharides of the plant cell wall that are made in the Golgi and secreted to the wall. The methylesterification of pectins is believed to be an important factor for the dynamic properties of the cell wall. Here, we report on a protein of unknown function discovered using an extensive proteomics analysis of cotton Golgi. Through bioinformatic analyses, we identified the ortholog of such protein, here named cotton Golgi-related 3 (CGR3) in Arabidopsis and found that it shares conserved residues with S-adenosylmethionine methyltransferases. We established that CGR3 is localized at the Golgi apparatus and that the expression of the CGR3 gene is correlated with that of several cell wall biosynthetic genes, suggesting a possible role of the protein in pectin modifications. Consistent with this hypothesis, immunofluorescence microscopy with antibodies for homogalacturonan pectins (HG) indicated that the cell walls of cgr3 knockout mutants and plants overexpressing CGR3 are decreased and increased in HG methylesterification, respectively. Our results suggest that CGR3 plays a role in the methylesterification of homogalacturonan in Arabidopsis.

A central question in cell biology is how the identity of organelles is established and maintained. Here, we report on GOLD36, an EMS mutant identified through a screen for partial displacement of the Golgi marker, ST-GFP, to other organelles. GOLD36 showed partial distribution of ST-GFP into a modified endoplasmic reticulum (ER) network, which formed bulges and large skein-like structures entangling Golgi stacks. GOLD36 showed defects in ER protein export as evidenced by our observations that, besides the partial retention of Golgi markers in the ER, the trafficking of a soluble bulk-flow marker to the cell surface was also compromised. Using a combination of classical mapping and next-generation DNA sequencing approaches, we linked the mutant phenotype to a missense mutation of a proline residue in position 80 to a leucine residue in a small endomembrane protein encoded by the gold36 locus (At1g54030). Subcellular localization analyses indicated that GOLD36 is a vacuolar protein and that its mutated form is retained in the ER. Interestingly also, a gold36 knock-out mutant mirrored the GOLD36 subcellular phenotype. These data indicate that GOLD36 is a protein destined to post-ER compartments and suggest that its export from the ER is a requirement to ensure steady-state maintenance of the organelle's organization and functional activity in relation to other secretory compartments. We speculate that GOLD36 may be a factor that is necessary for ER integrity because of its ability to limit deleterious effects of other secretory proteins on the ER., (© 2010 The Authors. Journal compilation © 2010 Blackwell Publishing Ltd.)

The majority of tumors, including melanoma, are phenotypically heterogeneous in that they contain various cell populations with differential expression of cell surface antigens such as CD133/Prominin-1. We have used fluorescence-activated cell sorting (FACS) technology to purify CD133(+) and CD133(-) cellular subsets from mouse melanoma models for high-quality total RNA practical for downstream applications such as expression profiling. Implementation of this strategy can lead to higher resolution of transcripts that are potentially important for the survival and functionality of one cancer cell population relative to another. Suboptimal extraction of RNA after FACS is common and can ultimately result in misinterpretations that impede the effective design of novel therapies. Here, we describe a number of methods that have been amenable to the successful isolation of high-quality total RNA after FACS of CD133(+) and CD133(-) mouse melanoma cell fractions.

Questions persist about the nature and number of cells with tumor-propagating capability in different types of cancer, including melanoma. In part, this is because identification and characterization of purified tumorigenic subsets of cancer cells has not been achieved to date. Here, we report tumor formation after injection of single purified melanoma cells derived from three novel mouse models. Tumor formation occurred after every injection of individual CD34+p75- melanoma cells, with intermediate rates using CD34-p75- cells, and rarely with CD34-p75+ cells. These findings suggest that tumorigenic melanoma cells may be more common than previously thought and establish that multiple distinct populations of melanoma-propagating cells (MPC) can exist within a single tumor. Interestingly, individual CD34-p75- MPCs could regenerate cellular heterogeneity after tumor formation in mice or multiple passages in vitro, whereas CD34+p75- MPCs underwent self-renewal only, showing that reestablishment of tumor heterogeneity is not always a characteristic of individual cells capable of forming tumors. Functionally, single purified MPCs were more resistant to chemotherapy than non-MPCs. We anticipate that purification of these MPCs may allow a more comprehensive evaluation of the molecular features that define tumor-forming capability and chemotherapeutic resistance in melanoma.

Small-interfering RNAs (siRNAs) from natural cis-antisense pairs derived from the 3'-coding region of the barley (Hordeum vulgare) CesA6 cellulose synthase gene substantially increase in abundance during leaf elongation. Strand-specific RT-PCR confirmed the presence of an antisense transcript of HvCesA6 that extends > or = 1230 bp from the 3' end of the CesA-coding sequence. The increases in abundance of the CesA6 antisense transcript and the 21-nt and 24-nt siRNAs derived from the transcript are coincident with the down-regulation of primary wall CesAs, several Csl genes, and GT8 glycosyl transferase genes, and are correlated with the reduction in rates of cellulose and (1 --> 3),(1 --> 4)-beta-D-glucan synthesis. Virus induced gene silencing using unique target sequences derived from HvCesA genes attenuated expression not only of the HvCesA6 gene, but also of numerous nontarget Csls and the distantly related GT8 genes and reduced the incorporation of D-(14)C-Glc into cellulose and into mixed-linkage (1 --> 3),(1 --> 4)-beta-D-glucans of the developing leaves. Unique target sequences for CslF and CslH conversely silenced the same genes and lowered rates of cellulose and (1 --> 3),(1 --> 4)-beta-D-glucan synthesis. Our results indicate that the expression of individual members of the CesA/Csl superfamily and glycosyl transferases share common regulatory control points, and siRNAs from natural cis-antisense pairs derived from the CesA/Csl superfamily could function in this global regulation of cell-wall synthesis.

In plants, differentiation of subdomains of the endoplasmic reticulum (ER) dedicated to protein export, the ER export sites (ERES), is influenced by the type of export-competent membrane cargo to be delivered to the Golgi. This raises a fundamental biological question: is the formation of transport intermediates at the ER for trafficking to the Golgi always regulated in the same manner? To test this, we followed the distribution and activity of two plant Sar1 isoforms. Sar1 is the small GTPase that regulates assembly of COPII (coat protein complex II) on carriers that transport secretory cargo from ER to Golgi. We show that, in contrast to a tobacco Sar1 isoform, the two Arabidopsis Sar1 GTPases were localised at ERES, independently of co-expression of Golgi-destined membrane cargo in tobacco cells. Although both isoforms labelled ERES, one was found to partition with the membrane fraction to a greater extent. The different distribution of fluorescent fusions of the two isoforms was influenced by the nature of an amino acid residue at the C-terminus of the protein, suggesting that the requirements for membrane association of the two GTPases are not equal. Furthermore, functional analyses based on the secretion of the bulk flow marker alpha-amylase indicated that over-expression of GTP-restricted mutants of the two isoforms caused different levels of ER export inhibition. These novel results indicate a functional heterogeneity among plant Sar1 isoforms.

Phosphate (Pi) is one of the least available plant nutrients in soils. It is associated with dynamic changes in carbon fluxes and several crucial processes that regulate plant growth and development. Pi levels regulate the expression of large number of genes including those involved in photosynthesis and carbon metabolism. Herein we show that sugar is required for Pi starvation responses including changes in root architecture and expression of phosphate starvation induced (PSI) genes in Arabidopsis. Active photosynthesis or the supplementation of sugar in the medium was essential for the expression of PSI genes under Pi limiting conditions. Expression of these genes was not only induced by sucrose but also detected, albeit at reduced levels, with other metabolizable sugars. Non-metabolizable sugar analogs did not induce the expression of PSI genes. Although sugar input appears to be down-stream of initial Pi sensing, it is absolutely required for the completion of the PSI signaling pathway. Altered expression of PSI genes in the hexokinase signaling mutant gin2 indicates that hexokinase-dependent signaling is involved in this process. The study provides evidence for requirement of sugars in PSI signaling and evokes a role for hexokinase in some components of Pi response mechanism.

Children with cholestatic liver diseases, in particular biliary atresia, may develop an acquired growth hormone (GH) resistance. This is characterized by normal GH secretion, reduced liver GH receptor (GHR) abundance, and reduced circulating insulin-like growth factor I (IGF-I). Consequences include linear growth failure, reduced muscle mass, and increased perioperative morbidity and mortality. However, the molecular basis for altered GH signaling in liver and skeletal muscle in cholestatic liver disease is not known. We hypothesized that reduced IGF-I expression in obstructive cholestasis would be associated with downregulation of the GHR and impaired phosphorylation of signal transducers and activators of transcription (STAT5). Body composition was determined in C57BL/6J male mice after bile duct ligation (BDL) relative to pair-fed (PF) and ad libitum-fed controls. GHR, STAT5, Sp3, and IGF-I expression and/or DNA binding were assessed using immunoblots, electrophoretic mobility shift assays, and/or real time RT-PCR. Fat-free mass was reduced in PF mice relative to ad libitum-fed controls. BDL led to a further reduction in fat mass and fat-free mass relative to PF controls. TNF-alpha was increased in liver and skeletal muscle of BDL mice. This was associated with reduced GH-dependent STAT5 activation and IGF-I RNA expression. GHR expression was reduced in BDL mice; in liver, this was associated with reduced Sp3 binding to a GHR gene promoter cis element. Wasting in murine obstructive cholestasis is due to combined effects of reduced caloric intake and biliary obstruction. GH resistance due to downregulation of GHR expression may be attributed primarily to the obstructive cholestasis; therapies that specifically increase GHR expression may restore GH signaling in this setting.

Background & Aims: Cytokines including tumor necrosis factor alpha (TNFalpha) may create a state of growth hormone (GH) resistance in Crohn's disease. Anabolic effects of GH are mediated via phosphorylation of the signal transducer and activator of transcription (STAT)5b transcription factor. Although GH resistance in other settings has been linked to a defect in janus kinase-STAT signaling, the molecular basis for GH resistance in colitis was not known. We hypothesized that the GH-induced phosphorylation of STAT5b would be impaired in colitis, and that TNFalpha blockade would restore GH signaling., Methods: Growth, body composition, and molecular regulators of GH signaling were determined in interleukin-10 null mice with chronic colitis and wild-type controls, +/- treatment with an anti-TNFalpha antibody., Results: Interleukin-10 null mice exhibited significant alterations in growth, body composition, and feed efficiency. Liver insulin-like growth factor 1 expression was reduced in colitic mice. This was associated with down-regulation of GH receptor (GHR) expression and impaired GH-dependent STAT5b activation. Down-regulation of GHR expression was associated with reduced nuclear abundance and DNA binding of the GHR gene-promoter transactivator, Sp3. TNFalpha down-regulated GHR abundance and prevented GH-induced tyrosine phosphorylation of STAT5 in rat hepatocytes in culture. TNFalpha neutralization up-regulated liver GHR abundance and restored GH activation of STAT5 and serum insulin-like growth factor 1 levels in colitic mice; this preceded improvements in weight gain and disease activity., Conclusions: GH resistance in experimental colitis is caused by down-regulation of GHR expression, thereby reducing GH-dependent STAT5 activation. TNFalpha blockade restores liver GH signaling and improves anabolic metabolism in this setting.

Extensins are cell wall hydroxyproline-rich glycoproteins that form covalent networks putatively involving tyrosyl and lysyl residues in cross-links catalyzed by one or more extensin peroxidases. The precise cross-links remain to be chemically identified both as network components in muro and as enzymic products generated in vitro with native extensin monomers as substrates. However, some extensin monomers contain variations within their putative cross-linking motifs that complicate cross-link identification. Other simpler extensins are recalcitrant to isolation including the ubiquitous P3-type extensin whose major repetitive motif, Hyp)(4)-Ser-Hyp-Ser-(Hyp)(4)-Tyr-Tyr-Tyr-Lys, is of particular interest, not least because its Tyr-Tyr-Tyr intramolecular isodityrosine cross-link motifs are also putative candidates for further intermolecular cross-linking to form di-isodityrosine. Therefore, we designed a set of extensin analogs encoding tandem repeats of the P3 motif, including Tyr --> Phe and Lys --> Leu variations. Expression of these P3 analogs in Nicotiana tabacum cells yielded glycoproteins with virtually all Pro residues hydroxylated and subsequently arabinosylated and with likely galactosylated Ser residues. This was consistent with earlier analyses of P3 glycopeptides isolated from cell wall digests and the predictions of the Hyp contiguity hypothesis. The tyrosine-rich P3 analogs also contained isodityrosine, formed in vivo. Significantly, these isodityrosine-containing analogs were further cross-linked in vitro by an extensin peroxidase to form the tetra-tyrosine intermolecular cross-link amino acid di-isodityrosine. This is the first identification of an inter-molecular cross-link amino acid in an extensin module and corroborates earlier suggestions that di-isodityrosine represents one mechanism for cross-linking extensins in muro.

Mrp3(Abcc3) is markedly induced following bile duct ligation (BDL) in the rat and in some human cholestatic liver diseases and is believed to ameliorate liver injury in this setting. Recently, the orphan nuclear receptor fetoprotein transcription factor/cholesterol-7alpha-hydroxylase promoter factor (CPF/FTF/Lrh-1) has been shown to activate Mrp3 expression. However, whether inflammatory cytokines or elevated bile acid levels increased Lrh-1/Mrp3 expression in obstructive cholestasis was not known. We hypothesized that induction of Mrp3 would be associated with Lrh-1 up-regulation and would require intact cytokine signaling. Male tumor necrosis factor (Tnf) receptor I (Tnfr-/-) mice and C57BLJ wild type (WT) controls were subjected to sham surgery or bile duct ligation. HepG2 cells were treated with bile acids or cytokines. Immunoblot assay and real time reverse transcriptase-PCR were used to determine expression of MRP3/Mrp3, CPF/Lrh-1, Mrp2, and Bsep. CPF/Lrh-1 DNA binding to the MRP3/Mrp3 promoter was assessed using electrophoretic mobility shift assay, and promoter activity was determined by luciferase assay. Total bile acids and lactate dehydrogenase were measured using colorimetric assays, and cytokine abundance was determined by enzyme-linked immunosorbent assay. Lrh-1 and Mrp3 were significantly induced after BDL in WT but not Tnfr-/- mice. This was associated with more severe hepatocellular necrosis in Tnfr-/- mice. Lrh-1 binding to the Mrp3 promoter increased after BDL in WT but not in Tnfr-/- mice. Tnfalpha treatment of HepG2 cells also up-regulated CPF and MRP3, increased CPF binding to the MRP3 promoter, and up-regulated MRP3 promoter activity. These results indicate that induction of Mrp3 after BDL is due to Tnfalpha-dependent up-regulation of Lrh-1. They provide strong evidence that induction of Mrp3 plays a significant role in hepatocyte protection during obstructive cholestasis.

Cytokines may cause an acquired growth hormone (GH) resistance in patients with inflammatory diseases. Anabolic effects of GH are mediated through activation of STAT5 transcription factors. We have reported that TNF-alpha suppresses hepatic GH receptor (GHR) gene expression, whereas the cytokine-inducible SH2-containing protein 1 (Cis)/suppressors of cytokine signaling (Socs) genes are upregulated by TNF-alpha and IL-6 and inhibit GH activation of STAT5. However, the relative importance of these mechanisms in inflammatory GH resistance was not known. We hypothesized that IL-6 would prevent GH activation of STAT5 and that this would involve Cis/Socs protein upregulation. GH +/- LPS was administered to TNF receptor 1 (TNFR1) or IL-6 null mice and wild-type (WT) controls. STAT5, STAT3, GHR, Socs 1-3, and Cis phosphorylation and abundance were assessed by using immunoblots, EMSA, and/or real time RT-PCR. TNF-alpha and IL-6 abundance were assessed by using ELISA. GH activated STAT5 in WT and TNFR1 or IL-6 null mice. LPS pretreatment prevented STAT5 activation in WT and TNFR1 null mice; however, STAT5 activation was preserved in IL-6 null mice. GHR abundance did not change with LPS administration. Inhibition of STAT5 activation by LPS was temporally associated with phosphorylation of STAT3 and upregulation of Cis and Socs-3 protein in WT and TNFR1 null mice; STAT3, Cis, and Socs-3 were not induced in IL-6 null mice. IL-6 inhibits hepatic GH signaling by upregulating Cis and Socs-3, which may involve activation of STAT3. Therapies that block IL-6 may enhance GH signaling in inflammatory diseases.