Improving transformation and regeneration efficiency in medicinal plants: Insights from other recalcitrant species.

Medicinal plants are integral to traditional medicine systems world-wide, being pivotal for human health. Harvesting plant material from natural environments, however, has led to species scarcity, prompting action to develop cultivation solutions that also aid conservation efforts. Biotechnological tools, specifically plant tissue culture and genetic transformation, offer solutions for sustainable, large-scale production and enhanced yield of valuable biomolecules. While these techniques are instrumental to the development of the medicinal plant industry, the challenge of inherent regeneration recalcitrance in some species to in vitro cultivation hampers these efforts. This review examines the strategies for overcoming recalcitrance in medicinal plants using a holistic approach, emphasising the meticulous choice of explants, e.g. embryonic/meristematic tissues; plant growth regulators, e.g. synthetic cytokinins; and use of novel regeneration-enabling methods to deliver morphogenic genes e.g. GRF/GIF chimeras and nanoparticles, which have been shown to contribute to overcoming recalcitrance barriers in agriculture crops. Furthermore, it highlights the benefit of cost-effective genomic technologies that enable precise genome editing and the value of integrating data-driven models to address genotype-specific challenges in medicinal plant research. These advances mark a progressive step towards a future where medicinal plant cultivation is not only more efficient and predictable but also inherently sustainable, ensuring the continued availability and exploitation of these important plants for current and future generations.

Does social participation make Thai psychologically abused elders happier? a stress-buffering effect hypothesis.

This study investigates the influence of psychological elder abuse on life satisfaction levels in Thailand. This study also analyses the stress-buffering effect of social participation on the life satisfaction levels of Thai mentally abused elderly. Elder abuse has been proven to dramatically reduce Thai elders' levels of life satisfaction as their function in society shrinks owing to ageism. As a result, individuals are more likely to lose their independence and status and be forced to rely on others, increasing the danger of abuse. Elder abuse has a more significant negative impact on life satisfaction levels among Thai older women. Nonetheless, the mentally abused elderly who participate in social activities are happier than those who do not. Thai elders who live with their daughters are more satisfied in life than those who do not, but living with adult offspring does not assist psychologically abused elders in escaping their psychological suffering.

Metabolomic analysis of methyl jasmonate treatment on phytocannabinoid production in Cannabis sativa.

Cannabis sativa is a multi-use and chemically complex plant which is utilized for food, fiber, and medicine. Plants produce a class of psychoactive and medicinally important specialized metabolites referred to as phytocannabinoids (PCs). The phytohormone methyl jasmonate (MeJA) is a naturally occurring methyl ester of jasmonic acid and a product of oxylipin biosynthesis which initiates and regulates the biosynthesis of a broad range of specialized metabolites across a number of diverse plant lineages. While the effects of exogenous MeJA application on PC production has been reported, treatments have been constrained to a narrow molar range and to the targeted analysis of a small number of compounds. Using high-resolution mass spectrometry with data-dependent acquisition, we examined the global metabolomic effects of MeJA in C. sativa to explore oxylipin-mediated regulation of PC biosynthesis and accumulation. A dose-response relationship was observed, with an almost two-fold increase in PC content found in inflorescences of female clones treated with 15 mM MeJA compared to the control group. Comparison of the inflorescence metabolome across MeJA treatments coupled with targeted transcript analysis was used to elucidate key regulatory components contributing to PC production and metabolism more broadly. Revealing these biological signatures improves our understanding of the role of the oxylipin pathway in C. sativa and provides putative molecular targets for the metabolic engineering and optimization of chemical phenotype for medicinal and industrial end-uses.

Heterologous production of Cannabis sativa-derived specialised metabolites of medicinal significance - Insights into engineering strategies.

Cannabis sativa L. has been known for at least 2000 years as a source of important, medically significant specialised metabolites and several bio-active molecules have been enriched from multiple chemotypes. However, due to the many levels of complexity in both the commercial cultivation of cannabis and extraction of its specialised metabolites, several heterologous production approaches are being pursued in parallel. In this review, we outline the recent achievements in engineering strategies used for heterologous production of cannabinoids, terpenes and flavonoids along with their strength and weakness. We provide an overview of the specialised metabolism pathway in C. sativa and a comprehensive list of the specialised metabolites produced along with their medicinal significance. We highlight cannabinoid-like molecules produced by other species. We discuss the key biosynthetic enzymes and their heterologous production using various hosts such as microbial and eukaryotic systems. A brief discussion on complementary production strategies using co-culturing and cell-free systems is described. Various approaches to optimise specialised metabolite production through co-expression, enzyme engineering and pathway engineering are discussed. We derive insights from recent advances in metabolic engineering of hosts with improved precursor supply and suggest their application for the production of C. sativa speciality metabolites. We present a collation of non-conventional hosts with speciality traits that can improve the feasibility of commercial heterologous production of cannabis-based specialised metabolites. We provide a perspective of emerging research in synthetic biology, allied analytical techniques and plant heterologous platforms as focus areas for heterologous production of cannabis specialised metabolites in the future.

A Model to Incorporate the bHLH Transcription Factor OsIRO3 within the Rice Iron Homeostasis Regulatory Network.

Iron (Fe) homeostasis in plants is governed by a complex network of regulatory elements and transcription factors (TFs), as both Fe toxicity and deficiency negatively impact plant growth and physiology. The Fe homeostasis network is well characterized in Arabidopsis thaliana and remains poorly understood in monocotyledon species such as rice (Oryza sativa L.). Recent investigation of the rice Fe homeostasis network revealed OsIRO3, a basic Helix-Loop-Helix (bHLH) TF as a putative negative regulator of genes involved in Fe uptake, transport, and storage. We employed CRISPR-Cas9 gene editing to target the OsIRO3 coding sequence and generate two independent T-DNA-free, loss-of-function iro3 mutants in rice cv. Nipponbare. The iro3 mutant plants had similar phenotype under nutrient-sufficient conditions and had stunted growth under Fe-deficient conditions, relative to a T-DNA free, wild-type control (WT). Under Fe deficiency, iro3 mutant shoots had reduced expression of Fe chelator biosynthetic genes (OsNAS1, OsNAS2, and OsNAAT1) and upregulated expression of an Fe transporter gene (OsYSL15), relative to WT shoots. We place our results in the context of the existing literature and generate a model describing the role of OsIRO3 in rice Fe homeostasis and reinforce the essential function of OsIRO3 in the rice Fe deficiency response.

You are my death: the shattered temporalities of zombie time.

This essay considers the relationship between the experience of life shortening chronic illness and the current COVID-19 crisis. Martin O'Brien uses his experience of living with cystic fibrosis to interrogate the temporal experience of living within a global pandemic. He returns to his concept of zombie time, the temporal experience of living longer than expected, in order to understand the presence of death as a way of life. The essay uses some of O'Brien's own art practices, and an analysis of his own sick, coughing body in order to think through what it means to live with cystic fibrosis during a pandemic, which mimics much of its features. O'Brien argues that we are currently occupying a widespread zombie time, which frames other people as carriers of death, and that we must find ways of being together in order to survive.

Multi-omics analysis on an agroecosystem reveals the significant role of organic nitrogen to increase agricultural crop yield.

Both inorganic fertilizer inputs and crop yields have increased globally, with the concurrent increase in the pollution of water bodies due to nitrogen leaching from soils. Designing agroecosystems that are environmentally friendly is urgently required. Since agroecosystems are highly complex and consist of entangled webs of interactions between plants, microbes, and soils, identifying critical components in crop production remain elusive. To understand the network structure in agroecosystems engineered by several farming methods, including environmentally friendly soil solarization, we utilized a multiomics approach on a field planted with Brassica rapa We found that the soil solarization increased plant shoot biomass irrespective of the type of fertilizer applied. Our multiomics and integrated informatics revealed complex interactions in the agroecosystem showing multiple network modules represented by plant traits heterogeneously associated with soil metabolites, minerals, and microbes. Unexpectedly, we identified soil organic nitrogen induced by soil solarization as one of the key components to increase crop yield. A germ-free plant in vitro assay and a pot experiment using arable soils confirmed that specific organic nitrogen, namely alanine and choline, directly increased plant biomass by acting as a nitrogen source and a biologically active compound. Thus, our study provides evidence at the agroecosystem level that organic nitrogen plays a key role in plant growth.

Are the current gRNA ranking prediction algorithms useful for genome editing in plants?

Introducing a new trait into a crop through conventional breeding commonly takes decades, but recently developed genome sequence modification technology has the potential to accelerate this process. One of these new breeding technologies relies on an RNA-directed DNA nuclease (CRISPR/Cas9) to cut the genomic DNA, in vivo, to facilitate the deletion or insertion of sequences. This sequence specific targeting is determined by guide RNAs (gRNAs). However, choosing an optimum gRNA sequence has its challenges. Almost all current gRNA design tools for use in plants are based on data from experiments in animals, although many allow the use of plant genomes to identify potential off-target sites. Here, we examine the predictive uniformity and performance of eight different online gRNA-site tools. Unfortunately, there was little consensus among the rankings by the different algorithms, nor a statistically significant correlation between rankings and in vivo effectiveness. This suggests that important factors affecting gRNA performance and/or target site accessibility, in plants, are yet to be elucidated and incorporated into gRNA-site prediction tools.

Life-span Extension Drug Interventions Affect Adipose Tissue Inflammation in Aging.

The National Institute on Aging (NIA)-sponsored Interventions Testing Program (ITP) has identified a number of dietary drug interventions that significantly extend life span, including rapamycin, acarbose, and 17-α estradiol. However, these drugs have diverse downstream targets, and their effects on age-associated organ-specific changes are unclear (Nadon NL, Strong R, Miller RA, Harrison DE. NIA Interventions Testing Program: investigating putative aging intervention agents in a genetically heterogeneous mouse model. EBioMedicine. 2017;21:3-4. doi:10.1016/j.ebiom.2016.11.038). Potential mechanisms by which these drugs extend life could be through their effect on inflammatory processes often noted in tissues of aging mice and humans. Our study focuses on the effects of three drugs in the ITP on inflammation in gonadal white adipose tissue (gWAT) of HET3 mice-including adiposity, adipose tissue macrophage (ATM) M1/M2 polarization, markers of cellular senescence, and endoplasmic reticulum stress. We found that rapamycin led to a 56% increase of CD45+ leukocytes in gWAT, where the majority of these are ATMs. Interestingly, rapamycin led to a 217% and 106% increase of M1 (CD45+CD64+CD206-) ATMs in females and males, respectively. Our data suggest rapamycin may achieve life-span extension in part through adipose tissue inflammation. Additionally, HET3 mice exhibit a spectrum of age-associated changes in the gWAT, but acarbose and 17-α estradiol do not strongly alter these phenotypes-suggesting that acarbose and 17- α estradiol may not influence life span through mechanisms involving adipose tissue inflammation.

Efficient sampling of shiitake-inoculated oak logs to determine the log-to-mushroom transfer factor of stable cesium.

BACKGROUND: Stable cesium (133Cs) naturally exists in the environment whereas recently deposited radionuclides (e.g., 137Cs) are not at equilibrium. Stable cesium has been used to understand the long-term behavior of radionuclides in plants, trees and mushrooms. We are interested in using 133Cs to predict the future transfer factor (TF) of radiocesium from contaminated logs to shiitake mushrooms in Eastern Japan. However, the current methodology to obtain a representative wood sample for 133Cs analysis involves mechanically breaking and milling the entire log (excluding bark) to a powder prior to analysis. In the current study, we investigated if sawdust obtained from cutting a log along its length at eight points is as robust but a faster alternative to provide a representative wood sample to determine the TF of 133Cs between logs and shiitake. METHODS: Oak logs with ready-to-harvest shiitake fruiting bodies were cut into nine 10-cm discs and each disc was separated into bark, sapwood and heartwood and the concentration of 133Cs was measured in the bark, sapwood, heartwood, sawdust (generated from cutting each disc) and fruiting bodies (collected separately from each disc), and the wood-to-shiitake TF was calculated. RESULTS: We found that the sawdust-to-shiitake TF of 133Cs did not differ (P = 0.223) compared to either the sapwood-to-shiitake TF or heartwood-to-shiitake TF, but bark did have a higher concentration of 133Cs (P < 0.05) compared to sapwood and heartwood. Stable cesium concentration in sawdust and fruiting bodies collected along the length of the logs did not differ (P > 0.05). DISCUSSION: Sawdust can be used as an alternative to determine the log-to-shiitake TF of 133Cs. To satisfy the goals of different studies and professionals, we have described two sampling methodologies (Methods I and II) in this paper. In Method I, a composite of eight sawdust samples collected from a log can be used to provide a representative whole-log sample (i.e., wood and bark), whereas Method II allows for the simultaneous sampling of two sets of sawdust samples-one set representing the whole log and the other representing wood only. Both methodologies can greatly reduce the time required for sample collection and preparation.

Adipose Tissue Senescence and Inflammation in Aging is Reversed by the Young Milieu.

Visceral adipose tissue (VAT) inflammation plays a central role in longevity and multiple age-related disorders. Cellular senescence (SEN) is a fundamental aging mechanism that contributes to age-related chronic inflammation and organ dysfunction, including VAT. Recent studies using heterochronic parabiosis models strongly suggested that circulating factors in young plasma alter the aging phenotypes of old animals. Our study investigated if young plasma rescued SEN phenotypes in the VAT of aging mice. With heterochronic parabiosis model using young (3 months) and old (18 months) mice, we found significant reduction in the levels of pro-inflammatory cytokines and altered adipokine profile that are protective of SEN in the VAT of old mice. These data are indicative of protection from SEN of aging VAT by young blood circulation. Old parabionts also exhibited diminished expression of cyclin-dependent kinase inhibitors (CDKi) genes p16 (Cdkn2a) and p21 (Cdkn1a/Cip1) in the VAT. In addition, when exposed to young serum condition in an ex vivo culture system, aging adipose tissue-derived stromovascular fraction cells produced significantly lower amounts of pro-inflammatory cytokines (MCP-1 and IL-6) compared to old condition. Expressions of p16 and p21 genes were also diminished in the old stromovascular fraction cells under young serum condition. Finally, in 3T3-preadipocytes culture system, we found reduced pro-inflammatory cytokines (Mcp-1 and Il-6) and diminished expression of cyclin-dependent kinase inhibitor genes in the presence of young serum compared to old serum. In summary, this study demonstrates that young milieu is capable of protecting aging adipose tissue from SEN and thereby inflammation.

Novel role of autophagy-associated Pik3c3 gene in gonadal white adipose tissue browning in aged C57/Bl6 male mice.

Adipose tissue dysfunction is associated with inflammation, metabolic syndrome and other diseases in aging. Recent work has demonstrated that compromised autophagy activity in aging adipose tissue promotes ER stress responses, contributing to adipose tissue and systemic inflammation in aging. Phosphatidylinositol 3-kinase catalytic subunit type 3 (Pik3c3) is an 887 amino acid lipid kinase that regulates intracellular membrane trafficking and autophagy activity. To address the mechanistic link between autophagy and ER stress response in aging adipose tissue, we generated a line of adipose tissue-specific Pik3c3 knock out (~mutant mice) with the Fabp4 (Fatty acid binding protein 4) promoter driven Cre recombinase system. We found elevated ER stress response signaling with reduced autophagy activity without any significant change on adiposity or glucose tolerance in early life of Pik3c3 mutant mice. Interestingly, middle- and old-aged mutant mice exhibited improved glucose tolerance (GTT) and reduced adiposity compared to age and sex-matched littermates. In addition, adipose tissue-specific Pik3c3 mutants display reduced expression of adiposity-associated genes with the signature of adipose tissue browning phenotypes in old age. Overall, the results suggest that altered adipose tissue characteristics due to autophagy inhibition early in life has beneficial effects that promote adipose tissue browning and improves glucose tolerance in late-life.

A unique role for galectin-9 in angiogenesis and inflammatory arthritis.

BACKGROUND: Galectin-9 (Gal-9) is a mammalian lectin secreted by endothelial cells that is highly expressed in rheumatoid arthritis synovial tissues and synovial fluid. Roles have been proposed for galectins in the regulation of inflammation and angiogenesis. Therefore, we examined the contribution of Gal-9 to angiogenesis and inflammation in arthritis. METHODS: To determine the role of Gal-9 in angiogenesis, we performed human dermal microvascular endothelial cell (HMVEC) chemotaxis, Matrigel tube formation, and mouse Matrigel plug angiogenesis assays. We also examined the role of signaling molecules in Gal-9-induced angiogenesis by using signaling inhibitors and small interfering RNA (siRNA). We performed monocyte (MN) migration assays in a modified Boyden chamber and assessed the arthritogenicity of Gal-9 by injecting Gal-9 into mouse knees. RESULTS: Gal-9 significantly increased HMVEC migration, which was decreased by inhibitors of extracellular signal-regulating kinases 1/2 (Erk1/2), p38, Janus kinase (Jnk), and phosphatidylinositol 3-kinase. Gal-9 HMVEC-induced tube formation was reduced by Erk1/2, p38, and Jnk inhibitors, and this was confirmed by siRNA knockdown. In mouse Matrigel plug assays, plugs containing Gal-9 induced significantly higher angiogenesis, which was attenuated by a Jnk inhibitor. Gal-9 also induced MN migration, and there was a marked increase in MN ingress when C57BL/6 mouse knees were injected with Gal-9 compared with the control, pointing to a proinflammatory role for Gal-9. CONCLUSIONS: Gal-9 mediates angiogenesis, increases MN migration in vitro, and induces acute inflammatory arthritis in mice, suggesting a novel role for Gal-9 in angiogenesis, joint inflammation, and possibly other inflammatory diseases.

Toll-like receptor 4 (TLR4) deficient mice are protected from adipose tissue inflammation in aging.

Adipose tissue (AT) inflammation is a central mechanism for metabolic dysfunction in both diet-induced obesity and age-associated obesity. Studies in diet-induced obesity have characterized the role of Fetuin A (Fet A) in Free Fatty Acids (FFA)-mediated TLR4 activation and adipose tissue inflammation. However, the role of Fet A & TLR4 in aging-related adipose tissue inflammation is unknown. In the current study, analysis of epidymymal fat pads of C57/Bl6 male mice, we found that, in contrast to data from diet-induced obesity models, adipose tissue from aged mice have normal Fet A and TLR4 expression. Interestingly, aged TLR4-deficient mice have diminished adipose tissue inflammation compared to normal controls. We further demonstrated that reduced AT inflammation in old TLR4-deficient mice is linked to impaired ER stress, augmented autophagy activity, and diminished senescence phenomenon. Importantly, old TLR4-deficient mice have improved glucose tolerance compared to age-matched wild type mice, suggesting that the observed reduced AT inflammation in aged TLR4-deficient mice has important physiological consequences. Taken together, our present study establishes novel aspect of aging-associated AT inflammation that is distinct from diet-induced AT inflammation. Our results also provide strong evidence that TLR4 plays a significant role in promoting aging adipose tissue inflammation.

Impaired autophagy activity is linked to elevated ER-stress and inflammation in aging adipose tissue.

Adipose tissue dysfunction in aging is associated with inflammation, metabolic syndrome and other diseases. We propose that impaired protein homeostasis due to compromised lysosomal degradation (micro-autophagy) might promote aberrant ER stress response and inflammation in aging adipose tissue. Using C57BL/6 mouse model, we demonstrate that adipose tissue-derived stromal vascular fraction (SVF) cells from old (18-20 months) mice have reduced expression of autophagy markers as compared to the younger (4-6 months) cohort. Elevated expressions of ER-stress marker CHOP and autophagy substrate SQSTM1/p62 are observed in old SVFs compared to young, when treated with either vehicle or with thapsigargin (Tg), an ER stress inducer. Treatment with bafilomycin A1 (Baf), a vacuolar-type H (+)-ATPase, or Tg elevated expressions of CHOP, and SQSTM1/p62 and LC-3-II, in 3T3-L1-preadipocytes. We also demonstrate impaired autophagy activity in old SVFs by analyzing increased accumulation of autophagy substrates LC3-II and p62. Compromised autophagy activity in old SVFs is correlated with enhanced release of pro-inflammatory cytokines IL-6 and MCP-1. Finally, SVFs from calorie restricted old mice (CR-O) have shown enhanced autophagy activity compared to ad libitum fed old mice (AL-O). Our results support the notion that diminished autophagy activity with aging contributes to increased adipose tissue ER stress and inflammation.

Epigenetic landscape of germline specific genes in the sporophyte cells of Arabidopsis thaliana.

In plants, the germline lineages arise in later stages of life cycle as opposed to animals where both male and female germlines are set aside early in development. This developmental divergence is associated with germline specific or preferential expression of a subset of genes that are normally repressed for the rest of plant life cycle. The gene regulatory mechanisms involved in such long-term suppression and short-term activation in plant germline remain vague. Thus, we explored the nature of epigenetic marks that are likely associated with long-term gene repression in the non-germline cells. We accessed available Arabidopsis genome-wide DNA methylation and histone modification data and queried it for epigenetic marks associated with germline genes: genes preferentially expressed in sperm cells, egg cells, synergid cells, central cells, antipodal cells or embryo sac or genes that are with enriched expression in two or more of female germline tissues. The vast majority of germline genes are associated with repression-related epigenetic histone modifications in one or more non-germline tissues, among which H3K9me2 and H3K27me3 are the most widespread repression-related marks. Interestingly, we show here that the repressive epigenetic mechanisms differ between male and female germline genes. We also highlight the diverse states of epigenetic marks in different non-germline tissues. Some germline genes also have activation-related marks in non-germline tissues, and the proportion of such genes is higher for female germline genes. Germline genes include 30 transposable element (TE) loci, to which a large number of 24-nt long small interfering RNAs were mapped, suggesting that these small RNAs take a role in suppressing them in non-germline tissues. The data presented here suggest that the majority of Arabidopsis gamete-preferentially/-enriched genes bear repressive epigenetic modifications or regulated by small RNAs.

PETAL LOSS, a trihelix transcription factor that represses growth in Arabidopsis thaliana, binds the energy-sensing SnRK1 kinase AKIN10.

Organogenesis in plants involves differential growth. Rapidly growing primordia are distinguished from the meristem and each other by slower growing boundaries. PETAL LOSS (PTL) is a trihelix transcription factor of Arabidopsis that represses growth in boundaries between newly arising sepals. To identify partners involved in this growth limitation, a young inflorescence cDNA library was screened by yeast two-hybrid technology with PTL as bait. The most frequent prey identified was AKIN10, the catalytic α-subunit of the Snf1-related kinase1 (SnRK1). Interaction was mapped to the C-terminal (non-kinase) half of AKIN10 and the N-terminal portion of PTL. Binding of PTL was specific to AKIN10 as there was little binding to the related AKIN11. The interaction was confirmed by co-immunoprecipitation in vitro. Fluorescently tagged products of 35S:YFP-AKIN10 and 35S:CFP-PTL also interacted when transiently expressed together in leaf cells of Nicotiana benthamiana. In this case, most of the cytoplasmic AKIN10 was preferentially moved to the nucleus where PTL accumulated, possibly because a nuclear export sequence in AKIN10 was now masked. During these experiments, we observed that AKIN10 could variably accumulate in the Golgi, shown by its co-localization with a tagged Golgi marker and through its dispersal by brefeldin A. Tests of phosphorylation of PTL by AKIN10 gave negative results. The functional significance of the PTL-AKIN10 interaction remains open, although a testable hypothesis is that AKIN10 senses lower energy levels in inter-sepal zones and, in association with PTL, promotes reduced cell division.

A key role for Fut1-regulated angiogenesis and ICAM-1 expression in K/BxN arthritis.

OBJECTIVES: Angiogenesis contributes to the pathogenesis of rheumatoid arthritis. Fucosyltransferases (Futs) are involved in angiogenesis and tumour growth. Here, we examined the role of Fut1 in angiogenesis and K/BxN serum transfer arthritis. METHODS: We examined Fut1 expression in human dermal microvascular endothelial cells (HMVECs) by quantitative PCR. We performed a number of angiogenesis assays to determine the role of Fut1 using HMVECs, Fut1 null (Fut1(-/-)), and wild type (wt) endothelial cells (ECs) and mice. K/BxN serum transfer arthritis was performed to determine the contribution of Fut1-mediated angiogenesis in Fut1(-/-) and wt mice. A static adhesion assay was implemented with RAW264.7 (mouse macrophage cell line) and mouse ECs. Quantitative PCR, immunofluorescence and flow cytometry were performed with Fut1(-/-) and wt ECs for adhesion molecule expression. RESULTS: Tumour necrosis factor-α induced Fut1 mRNA and protein expression in HMVECs. HMVECs transfected with Fut1 antisense oligodeoxynucleotide and Fut1(-/-) ECs formed significantly fewer tubes on Matrigel. Fut1(-/-) mice had reduced angiogenesis in Matrigel plug and sponge granuloma angiogenesis assays compared with wt mice. Fut1(-/-) mice were resistant to K/BxN serum transfer arthritis and had decreased angiogenesis and leucocyte ingress into inflamed joints. Adhesion of RAW264.7 cells to wt mouse ECs was significantly reduced when Fut1 was lacking. Fut1(-/-) ECs had decreased intercellular adhesion molecule-1 (ICAM-1) expression at mRNA and protein levels compared with wt ECs. ICAM-1 was also decreased in Fut1(-/-) arthritic ankle cryosections compared with wt ankles. CONCLUSIONS: Fut1 plays an important role in regulating angiogenesis and ICAM-1 expression in inflammatory arthritis.

Elevated Endoplasmic Reticulum Stress Response Contributes to Adipose Tissue Inflammation in Aging.

Adipose tissue inflammation has been linked to age-related metabolic diseases. However, the underlying mechanisms are poorly understood. Adipose tissue inflammation and insulin resistance in diet associated obesity has been correlated with aberrant endoplasmic reticulum (ER) stress. This study was undertaken to test our hypothesis that increased ER stress response contributes to age-associated adipose tissue inflammation. We found elevated ER stress response in adipose tissue of old (18-20 months) compared to young (4-6 months) mice. Elevated ER stress markers BIP (GRP78), CHOP, cleaved-ATF-6, phospho-IRE1α, and XBP-1 were observed in old compared to young adipose tissue stromal cells. Additionally, old adipose tissue stromal cells were more sensitive to an ER stress inducer, thapsigargin. Similar experiments with adipose tissue macrophages showed elevated Chop and Bip expression in old adipose tissue macrophages when induced with thapsigargin. Treatment of chemical chaperone 4-phenyle-butyric acid alleviated ER stress in adipose tissue stromal cells and adipose tissue macrophages and attenuated the production of IL-6 and MCP-1 by adipose tissue stromal cells, and TNF-α by adipose tissue macrophages from both young and old mice. Finally, old mice fed with 4-phenyle-butyric acid have reduced expression of ER stress and inflammatory cytokine genes. Our data suggests that an exaggerated ER stress response in aging adipose tissue contributes to age-associated inflammation that can be mitigated by treatment with chemical chaperones.

Functional domains of the PETAL LOSS protein, a trihelix transcription factor that represses regional growth in Arabidopsis thaliana.

PETAL LOSS (PTL) is a trihelix transcription factor that represses growth, especially between sepal primordia. As one of 30 trihelix proteins in Arabidopsis, it falls in the GT2 clade with duplicated trihelix DNA-binding domains and a long α-helical central domain. PTL orthologs occur in all angiosperm genomes examined except grasses, and sequence comparisons reveal that there are two further short conserved domains at each end. GT2 itself carries two nuclear localization sequences, but PTL has an additional nuclear localization sequence (NLS). We show that PTL can act as a transcriptional activator in yeast and in planta, with the latter tested by two different functional assays. Specific deletions revealed that the activation region is C-terminal. Site-directed mutagenesis of the DNA-binding domains has shown that a conserved tryptophan and two downstream acidic amino acids in the second trihelix, predicted to promote folding, are each required for PTL function. Also, three basic residues in the third helix, near the DNA interaction sites, support its function. PTL was found to dimerize in yeast. This was confirmed and extended by jointly expressing differentially tagged forms of PTL in a transient expression system in Nicotiana benthamiana leaves. Cytoplasmic PTL (with mutant NLS sequences) was carried into the nucleus upon binding with nuclear-localized PTL, providing each partner carried intact central domains. As this 90-amino acid domain is conserved in most trihelix family members, it seems likely that they all function in dimeric form.