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Saposin and its precursor prosaposin are endogenous proteins with neurotrophic and anti-apoptotic properties. Prosaposin or its analog prosaposin-derived 18-mer peptide (PS18) reduced neuronal damage in hippocampus and apoptosis in stroke brain. Its role in Parkinson’s disease (PD) has not been well characterized. This study aimed to examine the physiological role of PS18 in 6-hydroxydopamine (6-OHDA) cellular and animal models of PD. We found that PS18 significantly antagonized 6-OHDA -mediated dopaminergic neuronal loss and TUNEL in rat primary dopaminergic neuronal culture. In SH-SY5Y cells overexpressing the secreted ER calcium—monitoring proteins, we found that PS18 significantly reduced thapsigargin and 6-OHDA-mediated ER stress. The expression of prosaposin and the protective effect of PS18 were next examined in hemiparkinsonian rats. 6-OHDA was unilaterally administered to striatum. The expression of prosaposin was transiently upregulated in striatum on D3 (day 3) after lesioning and returned below the basal level on D29. The 6-OHDA-lesioned rats developed bradykinesia and an increase in methamphetamine-mediated rotation, which was antagonized by PS18. Brain tissues were collected for Western blot, immunohistochemistry, and qRTPCR analysis. Tyrosine hydroxylase immunoreactivity was significantly reduced while the expressions of PERK, ATF6, CHOP, and BiP were upregulated in the lesioned nigra; these responses were significantly antagonized by PS18. Taken together, our data support that PS18 is neuroprotective in cellular and animal models of PD. The mechanisms of protection may involve anti-ER stress.

We report the high-quality genome sequence of Tricholoma matsutake strain 2001, which was isolated from a mushroom fruiting body in South Korea. The genome has 80 contigs, a size of 162.6 Mb, and an N 50 value of 5,103,859 bp and will provide insight into the symbiotic association between T. matsutake and Pinus densiflora .

The CRISPR/Cas9 system has been used for genome editing in several plant species; however, there are few reports on its use in trees. Here, CRISPR/Cas9 was used to mutate a target gene in Populus alba × Populus glandulosa hybrid poplars. The hybrid poplar is routinely used in molecular biological studies due to the well-established Agrobacterium-mediated transformation method. A single guide RNA (sgRNA) with reported high mutation efficiency in other popular species was designed with a protospacer adjacent motif sequence for the phytoene desaturase 1 (PagPDS1) gene. The pHSE/Cas9-PagPDS1 sgRNA vector was delivered into hybrid poplar cells using Agrobacterium-mediated transformation. The transgenic plants were propagated and classified them into three groups according to their phenotypes. Among a total of 110 lines of transgenic hybrid poplars, 82 lines showed either an albino or a pale green phenotype, indicating around 74.5% phenotypic mutation efficiency of the PagPDS1 gene. The albino phenotypes were observed when the CRISPR/Cas9-mediated mutations in both PagPDS1 alleles in the transgenic plants. There was no off-target modification of the PagPDS2 gene, which has a potential sgRNA target sequence with two mismatches. The results confirmed that the sgRNA can specifically edit PagPDS1 rather than PagPDS2, indicating that CRISPR/Cas9-mediated genome editing can effectively induce target mutations in the hybrid poplar. This technique will be useful to improve tree quality in hybrid poplars (P. alba × P. glandulosa); for example, by enhancing biomass or stress tolerance.

Objective. Ankylosing spondylitis (AS) is a chronic inflammatory disease with obvious male preponderance. Males show more severe radiographic manifestations compared with females. This study aimed to evaluate the effects of sex and estrogen on the radiographic progression of AS. Methods. A total of 101 patients with AS were included in this study. All of the radiographs were scored using the modified Stoke AS Spine Score (mSASSS). Serum levels of 17β-estradiol (E2), dickkopf-1 (Dkk1), and leptin were detected by enzyme-linked immunosorbent assay. The generalized estimating equations model was used to evaluate factors associated with spinal radiographic progression. Results. The mean age at disease onset was 27.3±10.7 years, and 16 patients (15.8%) were female. In the multivariable analysis, body mass index (β-coefficient=0.12; p=0.047) and levels of Dkk1 (β-coefficient=-0.11; p＜0.001), and female (β-coefficient=-1.40; p=0.001) were associated with radiographic progression. Among male patients with AS, baseline C-reactive protein (β=0.11; p=0.005) and mSASSS (β=0.21; p=0.030) were also associated with radiographic progression. E2 and leptin levels were not significantly related to the radiographic progression. Conclusion. Although female patients were associated with less radiographic progression in AS, there was no significant relationship between serum estrogen level and radiographic progression. Results of current study suggests that genetic factors or other environmental factors associated with female may influence radiographic progression in patients with AS. (J Rheum Dis 2021;28:76-84)

Populus alba × P. glandulosa clone 84 K, derived from South Korea, is widely cultivated in China and used as a model in the molecular research of woody plants because of high gene transformation efficiency. Here, we combined 63-fold coverage Illumina short reads and 126-fold coverage PacBio long reads to assemble the genome. Due to the high heterozygosity level at 2.1% estimated by k-mer analysis, we exploited TrioCanu for genome assembly. The PacBio clean subreads of P. alba × P. glandulosa were separated into two parts according to the similarities, compared with the parental genomes of P. alba and P. glandulosa. The two parts of the subreads were assembled to two sets of subgenomes comprising subgenome A (405.31 Mb, from P. alba) and subgenome G (376.05 Mb, from P. glandulosa) with the contig N50 size of 5.43 Mb and 2.15 Mb, respectively. A high-quality P. alba × P. glandulosa genome assembly was obtained. The genome size was 781.36 Mb with the contig N50 size of 3.66 Mb and the longest contig was 19.47 Mb. In addition, a total of 176.95 Mb (43.7%), 152.37 Mb (40.5%) of repetitive elements were identified and a total of 38,701 and 38,449 protein-coding genes were predicted in subgenomes A and G, respectively. For functional annotation, 96.98% of subgenome A and 96.96% of subgenome G genes were annotated with public databases. This de novo assembled genome will facilitate systematic and comprehensive study, such as multi-omics analysis, in the model tree P. alba × P. glandulosa.

Plants have evolved defense mechanisms to overcome unfavorable climatic conditions. The growth and development of plants are regulated in response to environmental stress. In this study, we investigated the molecular and physiological characteristics of a novel gene PagSAP11 in hybrid poplar (Populus alba × Populus tremula var. glandulosa) under drought stress. PagSAP11, a stress-associated protein (SAP) family gene, encodes a putative protein containing an A20 and AN1 zinc-finger domain at its N- and C-termini, respectively. Knockdown of PagSAP11 transgenic poplars (SAP11-Ri) enhanced their tolerance to drought stress compared with wild type plants. Moreover, the RNAi lines showed increased branching of lateral shoots that led to a gain in fresh weight, even when grown in the living modified organism (LMO) field. In SAP11-Ri transgenic plants, the expression levels of genes involved in axillary bud outgrowth and cell proliferation such as DML10, CYP707A and RAX were increased while the DRM gene which involved in bud dormancy was down-regulated. Taken together, these results indicate that PagSAP11 represents a promising candidate gene for engineering trees with improved stress tolerance and growth during unfavorable conditions.

Unlike herbaceous plants, woody plants undergo volumetric growth (a.k.a. secondary growth) through wood formation, during which the secondary xylem (i.e., wood) differentiates from the vascular cambium. Wood is the most abundant biomass on Earth and, by absorbing atmospheric carbon dioxide, functions as one of the largest carbon sinks. As a sustainable and eco-friendly energy source, lignocellulosic biomass can help address environmental pollution and the global climate crisis. Studies of Arabidopsis and poplar as model plants using various emerging research tools show that the formation and proliferation of the vascular cambium and the differentiation of xylem cells require the modulation of multiple signals, including plant hormones, transcription factors, and signaling peptides. In this review, we summarize the latest knowledge on the molecular mechanism of wood formation, one of the most important biological processes on Earth.

Plants have evolved defense mechanisms to overcome unfavorable climatic conditions. The growth and development of plants are regulated in response to environmental stress. In this study, we investigated the molecular and physiological characteristics of a novel gene

Forests, comprising 31% of the Earth’s surface, play pivotal roles in regulating the carbon, water, and energy cycles. Despite being far less diverse than angiosperms, gymnosperms account for over 50% of the global woody biomass production. To sustain growth and development, gymnosperms have evolved the capacity to sense and respond to cyclical environmental signals, such as changes in photoperiod and seasonal temperature, which initiate growth (spring and summer) and dormancy (fall and winter). Cambium, the lateral meristem responsible for wood formation, is reactivated through a complex interplay among hormonal, genetic, and epigenetic factors. Temperature signals perceived in early spring induce the synthesis of several phytohormones, including auxins, cytokinins, and gibberellins, which in turn reactivate cambium cells. Additionally, microRNA-mediated genetic and epigenetic pathways modulate cambial function. As a result, the cambium becomes active during the summer, resulting in active secondary xylem (i.e., wood) production, and starts to become inactive in autumn. This review summarizes and discusses recent findings regarding the climatic, hormonal, genetic, and epigenetic regulation of wood formation in gymnosperm trees (i.e., conifers) in response to seasonal changes.

Stroke is a major cause of long-term disability world-wide. Limited pharmacological therapy has been used in stroke patients. Previous studies indicated that herb formula PM012 is neuroprotective against neurotoxin trimethyltin in rat brain, and improved learning and memory in animal models of Alzheimer’s disease. Its action in stroke has not been reported. This study aims to determine PM012-mediated neural protection in cellular and animal models of stroke. Glutamate-mediated neuronal loss and apoptosis were examined in rat primary cortical neuronal cultures. Cultured cells were overexpressed with a Ca++ probe (gCaMP5) by AAV1 and were used to examine Ca++ influx (Ca++i). Adult rats received PM012 before transient middle cerebral artery occlusion (MCAo). Brain tissues were collected for infarction and qRTPCR analysis. In rat primary cortical neuronal cultures, PM012 significantly antagonized glutamate-mediated TUNEL and neuronal loss, as well as NMDA-mediated Ca++i. PM012 significantly reduced brain infarction and improved locomotor activity in stroke rats. PM012 attenuated the expression of IBA1, IL6, and CD86, while upregulated CD206 in the infarcted cortex. ATF6, Bip, CHOP, IRE1, and PERK were significantly down-regulated by PM012. Using HPLC, two potential bioactive molecules, paeoniflorin and 5-hydroxymethylfurfural, were identified in the PM012 extract. Taken together, our data suggest that PM012 is neuroprotective against stroke. The mechanisms of action involve inhibition of Ca++i, inflammation, and apoptosis.

Gastrodia elata, an obligate mycoheterotrophic orchid, requires complete carbon and mineral nutrient supplementation from mycorrhizal fungi during its entire life cycle. Although full mycoheterotrophy occurs most often in the Orchidaceae family, no chromosome-level reference genome from this group has been assembled to date. Here, we report a high-quality chromosome-level genome assembly of G. elata, using Illumina and PacBio sequencing methods with Hi-C technique. The assembled genome size was found to be 1045 Mb, with an N50 of 50.6 Mb and 488 scaffolds. A total of 935 complete (64.9%) matches to the 1440 embryophyte Benchmarking Universal Single-Copy Orthologs were identified in this genome assembly. Hi-C scaffolding of the assembled genome resulted in 18 pseudochromosomes, 1008 Mb in size and containing 96.5% of the scaffolds. A total of 18,844 protein-coding sequences (CDSs) were predicted in the G. elata genome, of which 15,619 CDSs (82.89%) were functionally annotated. In addition, 74.92% of the assembled genome was found to be composed of transposable elements. Phylogenetic analysis indicated a significant contraction of genes involved in various biosynthetic processes and cellular components and an expansion of genes for novel metabolic processes and mycorrhizal association. This result suggests an evolutionary adaptation of G. elata to a mycoheterotrophic lifestyle. In summary, the genomic resources generated in this study will provide a valuable reference genome for investigating the molecular mechanisms of G. elata biological functions. Furthermore, the complete G. elata genome will greatly improve our understanding of the genetics of Orchidaceae and its mycoheterotrophic evolution.

Background To create an ideotype woody bioenergy crop with desirable growth and biomass properties, we utilized the viral 2A-meidated bicistronic expression strategy to express both PtrMYB3 (MYB46 ortholog of Populus trichocarpa, a master regulator of secondary wall biosynthesis) and PdGA20ox1 (a GA20-oxidase from Pinus densiflora that produces gibberellins) in wood-forming tissue (i.e., developing xylem). Results Transgenic Arabidopsis plants expressing the gene construct DX15::PdGA20ox1-2A-PtrMYB3 showed a significant increase in both stem fresh weight (threefold) and secondary wall thickening (1.27-fold) relative to wild-type (WT) plants. Transgenic poplars harboring the same gene construct grown in a greenhouse for 60 days had a stem fresh weight up to 2.6-fold greater than that of WT plants. In a living modified organism (LMO) field test conducted for 3 months of active growing season, the stem height and diameter growth of the transgenic poplars were 1.7- and 1.6-fold higher than those of WT plants, respectively, with minimal adverse growth defects. Although no significant changes in secondary wall thickening of the stem tissue of the transgenic poplars were observed, cellulose content was increased up to 14.4 wt% compared to WT, resulting in improved saccharification efficiency of the transgenic poplars. Moreover, enhanced woody biomass production by the transgenic poplars was further validated by re-planting in the same LMO field for additional two growing seasons. Conclusions Taken together, these results show considerably enhanced wood formation of our transgenic poplars, with improved wood quality for biofuel production.

Both anthocyanins and lignins are essential secondary metabolites in plant growth and development. Their biosynthesis is metabolically interconnected and diverges in the central metabolite 4-coumaroyl CoA of the phenylpropanoid pathway. Considerable progress has been made in understanding transcriptional regulation of genes involved in lignin and anthocyanin synthesis pathways, but the concerted regulation of these pathways is not yet fully understood. Here, we functionally characterized PtrMYB120, a R2R3-MYB transcription factor from Populus trichocarpa. Overexpression of PtrMYB120 in a hybrid poplar (i.e., 35S::PtrMYB120) was associated with increased anthocyanin (i.e., cyanidin 3-O-glucoside) accumulation and upregulation of anthocyanin biosynthetic genes. However, transgenic poplars with dominant suppression of PtrMYB120 function achieved by fusing the ERF-associated amphiphilic repression motif to PtrMYB120 (i.e., 35S::PtrMYB120-SRDX) had a dramatic decrease in not only anthocyanin but also Klason lignin content with downregulation of both anthocyanin and lignin biosynthetic genes. Indeed, 35S::PtrMYB120-SRDX poplars had irregularly shaped xylem vessels with reduced S-lignin content in stems, which was proportionally related to the level of the introduced PtrMYB120-SRDX gene. Furthermore, protoplast-based transcriptional activation assay using the PtrMYB120-GR system suggested that PtrMYB120 directly regulates genes involved in both anthocyanin and lignin biosynthesis, including chalcone synthase and ferulate-5 hydroxylase. Interestingly, the saccharification efficiency of line #6 of 35S::PtrMYB120-SRDX poplars, which had slightly reduced lignin content with a normal growth phenotype, was dramatically enhanced (>45%) by NaOH treatment. Taken together, our results suggest that PtrMYB120 functions as a positive regulator of both anthocyanin and lignin biosynthetic pathways and can be targeted to enhance saccharification efficiency in woody perennials.

Drought stress is one of the major environmental problems in the growth of crops and woody perennials, but it is getting worse due to the global climate crisis. XERICO, a RING (Really Interesting New Gene) zinc-finger E3 ubiquitin ligase, has been shown to be a positive regulator of drought tolerance in plants through the control of abscisic acid (ABA) homeostasis. We characterized a poplar (Populus trichocarpa) RING protein family and identified the closest homolog of XERICO called PtXERICO. Expression of PtXERICO is induced by both salt and drought stress, and by ABA treatment in poplars. Overexpression of PtXERICO in Arabidopsis confers salt and ABA hypersensitivity in young seedlings, and enhances drought tolerance by decreasing transpirational water loss. Consistently, transgenic hybrid poplars overexpressing PtXERICO demonstrate enhanced drought tolerance with reduced transpirational water loss and ion leakage. Subsequent upregulation of genes involved in the ABA homeostasis and drought response was confirmed in both transgenic Arabidopsis and poplars. Taken together, our results suggest that PtXERICO will serve as a focal point to improve drought tolerance of woody perennials.

Background: Ankylosing spondylitis is a male-predominant disease and previous study revealed that estrogen has an anti-inflammatory effect on the spondyloarthritis (SpA) manifestations in zymosan-induced SKG mice. This study aimed to evaluate the effect of selective estrogen receptor (ER) modulator lasofoxifene (Laso) on disease activity of SpA. Methods: Mice were randomized into zymosan-treated, zymosan + 17β-estradiol (E2)-treated, and zymosan + Laso-treated groups. Clinical arthritis scores were measured, and 18F-fluorodeoxyglucose (18F-FDG) small-animal positron emission tomography/computed tomography was performed to quantify joint inflammation. Bone mineral density (BMD) was measured. Fecal samples were collected and 16S ribosomal RNA gene sequencing was used to determine gut microbiota differences. Results: Both zymosan + E2-treated mice and zymosan + Laso-treated mice showed lower arthritis clinical scores and lower 18F-FDG uptake than zymosan-treated mice. BMD was significantly higher in zymosan + E2-treated mice and zymosan + Laso-treated mice than zymosan-treated mice, respectively. Fecal calprotectin levels were significantly elevated at 8 weeks after zymosan injection in zymosan-treated mice, but it was not significantly changed in zymosan + E2-treated mice and zymosan + Laso-treated mice. Gut microbiota diversity of zymosan-treated mice was significantly different from zymosan + E2-treated mice and zymosan + Laso-treated mice, respectively. There was no significant difference in gut microbiota diversity between zymosan + E2-treated mice and zymosan + Laso -treated mice. Conclusions: Selective ER modulator Laso inhibited joint inflammation and enhanced BMD in SKG mice, a model of SpA. Laso also affected the composition and biodiversity of gut microbiota. This study provides new knowledge regarding that selected SpA patients could benefit from selective ER modulator treatment.

Downregulating PagSAP1 expression increases salt tolerance in poplar and leads to the alterations in the regulation of genes involved in maintaining cellular ionic homeostasis. Plants have evolved defense mechanisms to cope with unfavorable climate conditions such as salinity, drought, and extreme temperatures. Such adaptive strategies include fine-tuning of stress-associated proteins (SAPs). Here, we investigated the molecular and physiological characteristics of the novel gene PagSAP1 from hybrid poplar (Populus alba × P. glandulosa) in response to salt stress. PagSAP1 encodes a putative protein containing an A20 and AN1 zinc-finger domain at its N- and C-terminus, respectively. Salt stress significantly downregulated the expression of PagSAP1 in roots, but not leaves, of poplar at the seven-leaf stage. Compared to control plants, knockdown (RNAi) transgenic poplar plants showed strong tolerance to salt stress, while overexpression (OX) of PagSAP1 increased salt sensitivity. Analysis of Ca2+, Na+, and K+ contents in roots and leaves following salt treatment (150 mM NaCl) revealed that RNAi plants accumulated more Ca2+ and K+, and less Na+ than PagSAP1 OX plants. The RNAi lines exhibited increased expression of genes involved in maintaining cellular ionic homeostasis across the plasma membrane, such as salt overly sensitive 3 (SOS3), SOS1, high-affinity K+ transporter 1 (HKT1), H+-ATPase, AAA-type ATPase, and Arabidopsis K+ channel 2 (AKT2). Taken together, these results indicate that PagSAP1 represents a promising candidate gene for engineering trees with improved salt tolerance, which would increase their suitability for planting in marginal lands, such as reclaimed areas.

JA는 병원균과 곤충에 대한 방어기작 뿐만 아니라 식물 노화에도 관여하는 식물 호르몬이다. Basic helix-loop-helix 전사인자인 MYC2는 JA의 신호전달반응의 핵심조절자 역할을 하는 것으로 알려져 있다. 본 연구에서는 현사시나무에서 MYC2 유전자를 분리하고 발현특성을 조사하였으며, 다양한 환경 스트레스에 대한 내성을 갖는 임목을 생산하기 위하여 MYC2를 과발현시킨 현사시나무를 개발하였다. 포트에 식재된 MYC2 과발현 현사시나무는 대조구에 비해 잎 노화 표현형이 지연되는 특징을 보였으며, 엽록소 손실이 적은 것으로 나타났다. 또한 가을의 온도 및 광 주기 조건에서 MYC2 과발현 현사시나무의 광화학 효율을 측정한 결과 대조구보다 높은 특징을 보였다. 따라서 현사시나무의 MYC2 유전자가 낙엽이 지는 가을 동안에도 지속적인 생장을 가능하게 하여 임목의 바이오매스를 증진시키는데 기여할 수 있을 것으로 판단된다.

Introduction Although there have been many efforts to diagnose functional dyspepsia (FD), an objective diagnosis is not easily made. Korean medicine (KM) doctors have attempted to study parameters for diagnosing FD in clinical studies. The aim of this study was to select diagnostic parameters of FD based on pattern identification in KM. Methods First, we searched the relevant literature with Korean authors, recorded in 7 databases, from their inception to May 2016. Second, we consulted expert opinion to extract the diagnostic parameters from a standard questionnaire of pattern identification for FD in KM. Finally, we selected the diagnostic parameters from literature review findings and expert consensus process. Results We selected data on diagnostic parameters (including medical imaging parameters, such as gastric emptying time, mapping of skin surface temperature, and geometry and color information of the upper abdomen; biosignal detection, such as pressure pain threshold, pressure pain changes, and pain reduction change after thermal stimulation; medical laboratory testing such as oral moisture on the buccal mucosa, submandibular salivary flow, and saliva pH; and a self-reporting standard questionnaire based on pain visual analogue scale, frequency, and duration of pain in the upper abdomen) of FD obtained through literature review and expert consensus. Conclusions This study is important because it presents the diagnostic parameters of FD based on pattern types in KM. This is a significant first step toward quantitative assessment for the development of a diagnostic device.

Caffeoyl shikimate esterase (CSE) has been shown to play an important role in lignin biosynthesis in plants and is, therefore, a promising target for generating improved lignocellulosic biomass crops for sustainable biofuel production. Populus spp. has two CSE genes (CSE1 and CSE2) and, thus, the hybrid poplar (Populus alba × P. glandulosa) investigated in this study has four CSE genes. Here, we present transgenic hybrid poplars with knockouts of each CSE gene achieved by CRISPR/Cas9. To knockout the CSE genes of the hybrid poplar, we designed three single guide RNAs (sg1–sg3), and produced three different transgenic poplars with either CSE1 (CSE1-sg2), CSE2 (CSE2-sg3), or both genes (CSE1/2-sg1) mutated. CSE1-sg2 and CSE2-sg3 poplars showed up to 29.1% reduction in lignin deposition with irregularly shaped xylem vessels. However, CSE1-sg2 and CSE2-sg3 poplars were morphologically indistinguishable from WT and showed no significant differences in growth in a long-term living modified organism (LMO) field-test covering four seasons. Gene expression analysis revealed that many lignin biosynthetic genes were downregulated in CSE1-sg2 and CSE2-sg3 poplars. Indeed, the CSE1-sg2 and CSE2-sg3 poplars had up to 25% higher saccharification efficiency than the WT control. Our results demonstrate that precise editing of CSE by CRISPR/Cas9 technology can improve lignocellulosic biomass without a growth penalty.

Adventitious buds were obtained from isolated cotyledons cultured on MS medium with various concentrations of 6-benzylamino purine (BA) and thidiazuron (TDZ). The highest numbers of adventitious buds were obtained on MS medium supplemented with 0.2 mg/L BA. Experimental culturing with half the petiole portion and half with the terminal segments were grown on MS medium contained with 0.2 mg/L BA. Frequency of the adventitious bud induction was variable accordingly to the type of cultured explants. Explants with the half petiole showed the highest adventitious bud induction rate (80%) compared to explants of half with terminal segment (20%). An elongated shoot from the buds and growth of advent roots were both possible on the 1/2 MS medium without a plant growth regulator. These results offer an effective way in which clonal propagation can be accomplished.

Background Ankylosing spondylitis (AS) is a male-predominant disease, and radiographic evidence of damage is also more severe in males. Estrogen modulates immune-related processes such as T cell differentiation and cytokine production. This study aimed to evaluate the effect of estrogen on the disease activity of spondyloarthritis (SpA). Methods The effects of estrogen on the development of arthritis were evaluated by performing ovariectomy and 17β-estradiol (E2) pellet implantation in zymosan-treated SKG mice. Clinical arthritis scores were measured, and 18F-fluorodeoxyglucose (18F-FDG) small-animal positron emission tomography/computed tomography performed to quantify joint inflammation. The expression of inflammatory cytokines in joint tissue was measured. Results E2-treated mice showed remarkable suppression of arthritis clinically and little infiltration of inflammatory cells in the Achilles tendon and intervertebral disc. 18F-FDG uptake was significantly lower in E2-treated mice than in sham-operated (sham) and ovariectomized mice. Expression of TNF, interferon-γ, and IL-17A was significantly reduced in E2-treated mice, whereas expression of sclerostin and Dickkopf-1 was increased in E2-treated mice compared with sham and ovariectomized mice. Conclusions Estrogen suppressed arthritis development in SKG mice, a model of SpA. Results of this study suggest that estrogen has an anti-inflammatory effect on the spondyloarthritis manifestations of the SKG arthritis model.

Monodehydroascorbate reductase (MDHAR) is an important enzyme that plays a role in the detoxification of reactive oxygen species (ROS) by maintaining reduced pool of ascorbate through recycling the oxidized form of ascorbate. In this study, we isolated a PagMDHAR1 gene from Populus alba × P. glandulosa , and investigated its expression characteristics. The PagMDHAR1 cDNA encodes a putative 434 amino acids containing FAD- and NAD(P)H-binding domains. Southern blot analysis indicated that a single nuclear gene encodes this enzyme. Northern hybridization analysis revealed that PagMDHAR1 is highly expressed in both suspension cells and flower tissues, while its expression levels were enhanced by drought, salt, cold, wounding and ABA. Therefore, PagMDHAR1 might be expressed in response to abiotic stress through the ABA-mediated signaling pathway in this poplar species, suggesting that the PagMDHAR1 plays an important role in the defense mechanisms against oxidative stress. Keywords

Aim To investigate the effects of Tubastatin A, a selective histone deacetylase-6 inhibitor, on synovial inflammation and joint destruction in a collagen antibody-induced arthritis (CAIA) mouse model. Methods Collagen antibody-induced arthritis mice were given daily intraperitoneal injections of various concentrations of Tubastatin A (0, 10, 50, 100 mg/kg). The clinical score and paw thickness were measured. Mice were sacrificed on day 15, and the expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1 and IL-6 in the serum were analyzed using enyme-linked immunosorbent assay (ELISA). Two pathologists independently measured the synovitis score. Micro-computed tomography (CT) scans of the joints were performed to quantify joint destruction. The expression of IL-6 from human fibroblast-like synoviocytes (FLSs) after incubation with various doses of Tubastatin A (0, 0.75, 1.5, 3 μmol/L) was measured using ELISA. Results The clinical arthritis score was significantly attenuated and paw thickness was lower in the group treated with 100 mg/kg Tubastatin A compared with those treated with vehicle alone. The synovitis score was significantly reduced in the 100 mg/kg Tubastatin A-treated group compared with the control group. Micro-CT showed that quantitative measures of joint destruction were significantly attenuated in the 100 mg/kg Tubastatin A-treated group compared with the control. The expression of IL-6 in the sera was lower in the mice treated with Tubastatin A compared with the control. The expression of IL-6 in human FLSs decreased dose-dependently after incubation with Tubastatin A without affecting cell viability. Conclusions Tubastatin A successfully ameliorated synovial inflammation and protected against joint destruction in CAIA mice, at least in part, by modulating IL-6 expression.

Plant senescence is one of the survival strategies to use limited nutrients efficiently during growth, development and adaptation. In this study, we isolated a gene (PagSEN1) homologous to SENESCENCE 1 from Populus alba ⨯ P. glandulosa. The PagSEN1 gene encodes a putative protein consisting of 243 amino acids containing a rhodanese domain. Southern blot analysis suggested that two copies of the PagSEN1 gene are present in the poplar genome. We characterized its transcriptional expression under various conditions mimicking senescence and environmental stresses. The PagSEN1 was expressed most strongly in mature leaves but most weakly in roots. The gene was significantly up-regulated by treatments with mannitol, NaCl, ABA and JA, but not by cold, SA and GA3. These results indicate that PagSEN1 is involved in senescence response induced by environmental stresses.

Basic leucine zipper (bZIP) protein is a regulatory transcription factor that plays crucial roles in growth, development and stress response of plant. In this study, we isolated a PagbZIP1 gene that belonged to Group SE3 of bZIP from Populus alba × P. glandulosa, and investigated its expressional characteristics. The PagbZIP1 is 844 base pairs long and encodes a putative 144-amino-acid protein with an expected molecular mass of 16.6 kDa. The PagbZIP1 has two conserved domains including the basic and leucine zipper portions. Southern blot analysis revealed that two copies of the gene are presented in the poplar genome. PagbZIP1 was specifically expressed in the root and suspension cells. Moreover, the expression of PagbZIP1 was induced by drought, salt, cold and ABA. Therefore, our results indicated that PagbZIP1 might be expressed in response to abiotic stress through the ABA-mediated signaling pathway in poplar.

Patatin-liked phospholipase A (pPLAs) are major lipid acyl hydrolases that participate in various biological functions in plant growth and development. Previously, a ginseng-derived pPLAIII homolog was reported to reduce lignin content in Arabidopsis. This led us to evaluate its possible usefulness as a biomass source in wood plant. Herein, we report that there are six members in the pPLAIII gene family in poplar. Overexpression of pPLAIIIβ derived from ginseng resulted in a reduced plant height with radially expanded stem growth in hybrid poplars. Compared with the wild type (WT), the chlorophyll content was increased in the overexpression poplar lines, whereas the leaf size was smaller. The secondary cell wall structure in overexpression lines was also altered, exhibiting reduced lignification in the xylem. Two transcription factors, MYB92 and MYB152, which control lignin biosynthesis, were downregulated in the overexpression lines. The middle xylem of the overexpression line showed heavy thickening, making it thicker than the other xylem parts and the WT xylem, which rather could have been contributed by the presence of more cellulose in the selected surface area. Taken together, the results suggest that PgpPLAIIIβ plays a role not only in cell elongation patterns, but also in determining the secondary cell wall composition.

Objectives To investigate the effects of benzo[a]pyrene (B[a]P), a major toxic component of cigarette smoke, on the expression of Slug and to determine the effect of B[a]P/Slug on the invasive properties of rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS). Method The expression of Slug was measured by real-time PCR following the stimulation of FLS with different concentrations of B[a]P or EGF. The phosphorylation of the key enzymes in the signaling pathway was analyzed by western blots. Inhibitors of PI3K/Akt/mTOR pathway were used to confirm the critical pathway for Slug expression. An in vitro cell invasion assay was performed using RA FLS treated with Slug cDNA, Slug small interference RNA, or control. Results Slug expression increased significantly following treatment with B[a]P or EGF in a dose-dependent manner. The stimulation of FLS with B[a]P or EGF induced the phosphorylation of Akt kinase, but not in ERK, JNK and p38. The Slug mRNA expression induced by B[a]P and EGF decreased significantly following the treatment with PI3K/Akt/mTOR inhibitors. Slug overexpression using Slug cDNA upregulated the invasive function of FLS, and Slug depletion using siRNA showed the opposite effect compared with the control. In addition, the stimulation with B[a]P increased the invasive function of the control siRNA-treated FLS but not in the Slug siRNA-treated FLS. Conclusion Our data showed that B[a]P regulates the invasive properties of RA FLS through Slug expression. This mechanism may provide a novel molecular link underlying the association between smoking and increased radiographic progression in RA.

The gene expression profiles of hybrid poplar (Populus alba × Populus tremula var. glandulosa) cells in suspension culture after exposure to salinity (NaCl) induced stress were examined by constructing two suppression subtractive hybridization (SSH) libraries. cDNA from non-treated cells was used as a driver and cDNA samples from cell suspension cultures exposed to 150 mM NaCl for 2 or 10 h were used as testers. Randomly selected clones from each SSH library were sequenced and 727 high-quality expressed sequence tags (ESTs) were obtained and analyzed. Four novel ESTs were identified. Between the two libraries, 542 unique SSH clones were selected for placement on a cDNA microarray. In total, 18 differentially expressed genes were identified with 4 and 12 genes being significantly differentially expressed 2 and 10 h after the treatment, respectively. Genes related to metabolism and protein synthesis and several genes whose protein products are implicated in salt or other abiotic stress-related responses were expressed in the salt-stressed cells.

The aim of this study is to compare the expression of TIM-3 from CD4+ T cells from rheumatoid arthritis (RA) patients and healthy controls and to evaluate the effect of galectin-9 (Gal-9) on apoptosis of CD4+ T cells in these patients. CD4+ T cells from RA patients and healthy controls were isolated from peripheral blood mononuclear cells and were activated. The expression of TIM-3 mRNA in CD4+ T cells was measured using real-time polymerase chain reaction. CD4+ T cells were activated in the presence of graded doses of Gal-9 or control, and Gal-9-induced cytotoxicity and apoptotic activity of CD4+ T cells were analyzed using MTT assays and annexin-V staining, respectively. TIM-3 mRNA expression was significantly lower in CD4+ T cells from RA patients compared with those in healthy controls (p = 0.028). CD4+ T cell survival as measured by MTT assay when incubated with Gal-9 (15 nM) was significantly higher in RA patients than in healthy controls (p = 0.002). Apoptotic activity of CD4+ T cells from healthy controls as measured by annexin staining increased with graded doses of Gal-9 (0 nM vs. 30 nM, 0 nM vs. 90 nM, p = 0.016 each). However, apoptotic activity of CD4+ T cells from RA patients did not change despite the stimulation with Gal-9. Gal-9-mediated apoptosis of CD4+ T cells is dysfunctional in RA patients. Blunted Gal-9-mediated apoptosis may be exerted through underexpression of TIM-3 that negatively regulates Th1 response. Our data suggest that TIM-3 and its interaction with Gal-9 may play an important role in the pathogenesis of RA and may represent a potential therapeutic target.

Cancerous inhibitor of protein phosphatase 2A (CIP2A) is a recently identified oncoprotein that leads to cellular proliferation in cancer cells. We aim to investigate CIP2A expression in fibroblast-like synoviocytes (FLS) and its association with the histopathological grade of synovitis and the invasive function of FLS in rheumatoid arthritis (RA). CIP2A protein expression was measured in 8 RA FLS and 8 OA FLS using Western blot analysis. CIP2A mRNA expression from 19 RA FLS and 7 OA FLS was measured using real-time PCR. Synovitis score of RA FLS-matched synovial tissues was semiquantitatively measured by two independent pathologists. An in vitro cell invasion assay was performed using RA FLS treated with CIP2A small interfering RNA (siRNA) or with control vector. Western blot analysis showed that CIP2A is more frequently overexpressed in RA FLS compared with OA FLS. CIP2A mRNA expression was higher in RA FLS compared with those in OA FLS, but did not reach statistical significance (P = 0.076). In RA, total synovitis score was strongly correlated with FLS CIP2A mRNA expression (rs = 0.849, P = 0.043). TNF-α treatment induced a robust increase in the invasive function of control FLS (P = 0.0021), but no significant effect was observed in CIP2A siRNA-treated FLS. Our data demonstrate that CIP2A expression is closely associated with the histopathological score of synovitis and invasive function of FLS in RA. These results suggest that CIP2A may play a critical role in the destructive process in RA and warrant further investigation of CIP2A as a therapeutic target.

T-cell immunoglobulin- and mucin-domain-containing molecule 3 (TIM-3) is a novel transmembrane protein that is involved in the regulation of T-helper 1 (Th1)-cell-mediated immunity. This study was undertaken to investigate the expressions of TIM-3 and its ligand galectin 9 (Gal-9) with respect to disease activity in rheumatoid arthritis (RA).Blood was collected from 39 RA patients and 31 healthy controls. Blood leucocyte TIM-3 and Gal-9 mRNA levels and RA disease activity were determined. Synovial tissue (ST) from five RA patients and five osteoarthritis (OA) patients were examined for TIM-3 mRNA expression and were also analysed for TIM-3 by immunohistology.TIM-3 mRNA expression was significantly higher in the ST of RA patients than in the ST of OA patients. TIM-3 was expressed in the synovial sublining area in ST of RA patients but not in OA ST. TIM-3 mRNA expression from peripheral blood mononuclear cells (PBMCs) of RA patients was negatively correlated with the 28-joint Disease Activity Score (DAS28). Gal-9 mRNA level in PBMCs of RA patients was higher than in healthy controls, and was significantly higher in patients with low disease activity compared to those with moderate to high disease activity. Gal-9 mRNA expression in PBMCs of RA patients was positively correlated with forkhead box P3 (FoxP3) mRNA expression.TIM-3 and its interaction with Gal-9 are closely associated with RA disease activity and may play an important role in the pathogenesis of RA. In addition to the negative regulatory effect of Gal-9 mediated through the TIM-3-Gal-9 pathway, Gal-9 may exert its suppressive effect on RA disease activity by modulation of regulatory T (Treg) cells.

Survivin belongs to the family of inhibitor of apoptosis proteins and plays an important role in the hyperplastic growth of tissues and tumors. In this study, we assessed the expression of survivin in rheumatoid synovial fluids (SF) and synovial tissues (ST) of rheumatoid arthritis (RA) patients in order to investigate the role of extracellular survivin in the pathogenesis of RA. The survivin level from SF was significantly higher in RA patients (n = 38) than in osteoarthritis patients (n = 18; 10.68 ± 2.76 vs. 1.0 ± 0.56 pg/ml, p = 0.02). In addition, SF survivin level was higher in erosive RA patients (n = 23) than in non-erosive RA patients (n = 15; 15.26 ± 4.26 vs. 4.47 ± 1.12 pg/ml, p = 0.05). SF survivin level in RA was positively correlated with disease activity score 28, but did not reach statistical significance (r = 0.309, p = 0.07). RA SF survivin level was also positively correlated with peripheral blood leukocyte counts (r = 0.443, p = 0.005). The immunohistochemical staining and Western blot analysis revealed survivin expression in the ST and fibroblast-like synoviocytes of RA patients, respectively. These findings suggest that extracellular survivin may be produced from rheumatoid FLS and may play an important role in the destructive RA process.

Osmotic stress induces changes in the expression of various genes including those associated with drought tolerance, cell wall metabolism and defense. We isolated 852 cDNA clones, the expression of which is induced by osmotic stress, from cells of a hybrid poplar (Populus alba x Populus tremula var. glandulosa) by suppression subtractive hybridization after mannitol treatment. We examined how stress affected their expression using cDNA microarray analysis, which identified 104 genes significantly up-regulated by osmotic stress. These include genes with functions related to transcription, signal transduction, cell wall metabolism and defense. Other gene transcripts encoding cysteine protease and aquaporin are also up-regulated during osmotic stress. The function of about one-third of the genes in poplar cells that were significantly up-regulated by stress is not known, suggesting that the cell suspension may offer an opportunity of finding novel genes otherwise never expressed and that we still need more information at the molecular level.

Mutations of nucleophosmin gene (NPM1) are known to be related to good prognosis in AML patients lacking FLT3 internal tandem duplication (FLT3-ITD). Recently, NPM1 mutations other than type A were reported, but their clinical significance is not well known. Retrospective medical record review of 106 de novo AML patients lacking FLT3-ITD, who received induction chemotherapy from three centers in Korea between 1997 and 2007, was performed. Direct sequencing of NPM1 and RT-PCR for FLT3-ITD was performed on genomic DNA derived from blood samples of patients before induction chemotherapy for detection of mutations. NPM1 mutation was detected in 18 patients, where 13 were type A mutants and 5 were non-type A mutants. CR, CR1-D and OS was not different according to NPM1 mutational status overall. But, non-type A NPM1 mutation was related to shorter CR1-D when compared with NPM1 wild types and NPM1 type A mutation (p = 0.004). OS was shorter in non-type A mutants when compared with NPM1 wild-type patients and NPM1 type A mutants (p = 0.001). The type of mutation of NPM1 is important for prognosis in de novo AML lacking FLT3-ITD. Non-A type NPM1 mutation is a poor prognostic factor.