Endoplasmic reticulum (ER) stress-induced podocyte apoptosis plays a critical role in the development of diabetic nephropathy (DN). Here, we tested the hypothesis that suppression of PERK-ATF4-CHOP pathway by Astragaloside IV (AS-IV) is associated with inhibition of ER stress-induced podocyte apoptosis in streptozotocin (STZ)-induced diabetic rats.
Despite the abundance of phosphorus in soil, very little is available as phosphate (Pi) for plants. Plants often experience low Pi (LP) stress. Intensive studies have been conducted to reveal the mechanism used by plants to deal with LP; however, Pi sensing and signal transduction pathways are not fully understood. Using in-gel kinase assays, we determined the activities of MPK3 and MPK6 in Arabidopsis thaliana seedlings under both LP and Pi-sufficient (Murashige and Skoog, MS) conditions. Using MKK9 mutant transgenic and crossed mutants, we analyzed the functions of MPK3 and MPK6 in regulating Pi responses of seedlings. The regulation of Pi responses by downstream components of MKK9-MPK3/MPK6 was also screened. LP treatment activated MPK3 and MPK6. Under both LP and MS conditions, mpk3 and mpk6 seedlings took up and accumulated less Pi than the wild-type; activation of MKK9-MPK3/MPK6 in transgenic seedlings induced the transcription of Pi acquisition-related genes and enhanced Pi uptake and accumulation, whereas its activation suppressed the transcription of anthocyanin biosynthetic genes and anthocyanin accumulation; WRKY75 was downstream of MKK9-MPK3/MPK6 when regulating the accumulation of Pi and anthocyanin, and the transcription of Pi acquisition-related and anthocyanin biosynthetic genes. These results suggest that the MKK9-MPK3/MPK6 cascade is part of the Pi signaling pathway in plants.
The transcription factor, SNAI2 is an inducer of the epithelial to mesenchymal transition (EMT) which mediates cell migration during development and tumor invasion. SNAI2 can also promote the generation of mammary epithelial stem cells from differentiated luminal cells when overexpressed. How SNAI2 regulates these critical and diverse functions is unclear. Here we show that the levels of SNAI2 expression are important for epidermal cell fate decisions. The expression of SNAI2 was found to be enriched in the basal layer of the interfollicular epidermis where progenitor cells reside and extinguished upon differentiation. Loss of SNAI2 resulted in premature differentiation whereas gain of SNAI2 expression inhibited differentiation. SNAI2 controls the differentiation status of epidermal progenitor cells by binding to and repressing the expression of differentiation genes with increased binding leading to further transcriptional silencing. Thus, the levels of SNAI2 binding to genomic targets determines the differentiation status of epithelial cells with increased levels triggering EMT and dedifferentiation, moderate (physiological) levels promoting epidermal progenitor function, and low levels leading to epidermal differentiation. Stem Cells 2014.
High-amylose cereal endosperm is rich in heterogeneous starch granules. In this paper, we investigated the morphology, structure and gelatinization properties of high-amylose maize endosperm starch. Starch had individual, aggregate and elongated heterogeneous granules. Most of individual granules were round with small size and had one central hilum. Aggregate and elongated granules consisted of many subgranules with central hila, and had irregular and rod/filamentous shapes, respectively. Iodine stained starch granules showed five types of polarization colors: blue, purple, fuchsia, dark red, and interior dark blue and exterior brown. Most of individual and aggregate granules had the color of dark red, that of elongated granules the color of interior dark blue and exterior brown. Amylose was mainly distributed in the hilum region and the circumference of starch granules. Aggregate and elongated granules had higher amylose content than individual granules. Elongated and individual granules had the highest and the lowest gelatinization resistance among high-amylose maize heterogeneous starch granules, respectively.
Podocyte loss and dysfunction play key role during the development of diabetic nephropathy (DN). The aim of this study was to observe the protective effects of astragaloside IV on podocyte in diabetic rats and explore its mechanisms preliminary. Healthy male Sprague-Dawley (SD) rats were randomized into normal control group, diabetic nephropathy group and diabetic nephropathy with AS-IV treatment group. DN was induced by intraperitoneal injection of streptozotocin (STZ). AS-IV treatment started 2 weeks before STZ injection and lasted 14 weeks. 24h Urinary proteins were measured 4, 8 and 12 weeks after STZ injection. Body weight, blood glucose, blood urea nitrogen (BUN), creatinine (Cr), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured 12 weeks after STZ injection. Renal pathology, podocyte morphological changes, podocyte density, protein and mRNA expression of integrin ?3, integrin ?1 and integrin-linked kinase (ILK) were detected by histopathology, electron microscopy, immunohistochemistry, western blot and real-time PCR, respectively. Hyperglycemia, proteinuria, mesangial expansion and podocyte loss, increased protein expression of ILK and decreased protein expression of integrin ?3 and integrin ?1 were detected in diabetic rats. AS-IV treatment ameliorated podocyte loss, renal histopathology and podocyte foot process effacement, decreased proteinuria, partially restored protein expression of integrin ?3, integrin ?1 and ILK. These findings suggested that AS-IV may protect podocyte and ameliorate diabetic nephropathy by inhibiting the expression of ILK and restoring the expression of integrin ?3?1 in diabetic rats.
Mutating or inhibiting genes encoding starch branching enzymes (SBEs) can increase the amylose content (AC) of cereals. We analyzed endosperm starches from three rice cultivars with different ACs and from transgenic lines derived from them. The transgenic lines had simultaneously inhibited SBE I and IIb genes. Compared with the starch from their wild-type parents, the starch from transgenic lines showed significantly increased apparent ACs and lamella size and decreased relative crystallinity, double helix content, and lamellar peak scattering intensity, and altered short-range ordered structure in the external region. These changes were more prominent in the line derived from the high-AC cultivar than in those derived from waxy and low-AC cultivars. Inhibiting both SBE I and IIb changed the crystalline structure of starch from A-type to CA-type in lines derived from waxy and low-AC cultivars, and from A-type to C-type in that derived from the high-AC cultivar.
Cell fate commitment during development is achieved through the expression of lineage-specific transcription factors. Recent studies have suggested that the expression of combinations of these lineage-specific transcription factors can convert adult somatic cells from one type to another. Here we report that the combination of p63, a master regulator of epidermal development and differentiation, and KLF4, a regulator of epidermal differentiation, is sufficient to convert dermal fibroblasts to a keratinocyte phenotype. Induced keratinocytes (KCs) expressed KC-specific proteins and had a transcriptome similar to KCs. Reprogramming to a KC phenotype was rapid and efficient with a vast majority of cells morphologically resembling and expressing KC-specific genes within a week of p63 and KLF4 transduction. Furthermore, p63 and KLF4 are capable of inducing a KC phenotype even in a cancerous cell line, highlighting their importance for epidermal specification. The robustness of the conversion process also allows the use of this as a model system to study the mechanisms of reprogramming.Journal of Investigative Dermatology advance online publication, 12 September 2013; doi:10.1038/jid.2013.327.
Accumulating evidence suggests that inflammatory processes are involved in the development of diabetic nephropathy (DN). However, there are no effective interventions for inflammation in the diabetic kidneys. Here, we tested the hypothesis that Astragaloside IV(AS-IV), a novel saponin purified from Astragalus membranaceus (Fisch) Bge, ameliorates DN in streptozotocin (STZ)-induced diabetic rats through anti-inflammatory mechanisms. Diabetes was induced with STZ (65 mg/kg) by intraperitoneal injection in rats. Two weeks after STZ injection, rats were divided into three groups (n=8/each group), namely, diabetic rats, diabetic rats treated with AS-IV at 5 and 10 mgkg(-1)d(-1), p.o., for 8 weeks. The normal rats were chosen as nondiabetic control group (n=8). The rats were sacrificed 10 weeks after induction of diabetes. AS-IV ameliorated albuminuria, renal histopathology and podocyte foot process effacement in diabetic rats. Renal NF-?B activity, as wells as protein and mRNA expression were increased in diabetic kidneys, accompanied by an increase in mRNA expression and protein content of TNF-?, MCP-1 and ICAM-1 in kidney tissues. The ?1-chain type IV collagen mRNA was elevated in the kidneys of diabetic rats. All of these abnormalities were partially restored by AS-IV. AS-IV also decreased the serum levels of TNF-?, MCP-1 and ICAM-1 in diabetic rats. These findings suggest that AS-IV, a novel anti-inflammatory agent, attenuated DN in rats through inhibiting NF-?B mediated inflammatory genes expression.
Sle1a.1 is part of the Sle1 susceptibility locus, which has the strongest association with lupus nephritis in the NZM2410 mouse model. In this study, we show that Sle1a.1 results in the production of activated and autoreactive CD4(+) T cells. Additionally, Sle1a.1 expression reduces the peripheral regulatory T cell pool, as well as induces a defective response of CD4(+) T cells to the retinoic acid expansion of TGF-?-induced regulatory T cells. At the molecular level, Sle1a.1 corresponds to an increased expression of a novel splice isoform of Pbx1, Pbx1-d. Pbx1-d overexpression is sufficient to induce an activated/inflammatory phenotype in Jurkat T cells and to decrease their apoptotic response to retinoic acid. PBX1-d is expressed more frequently in the CD4(+) T cells from lupus patients than from healthy controls, and its presence correlates with an increased central memory T cell population. These findings indicate that Pbx1 is a novel lupus susceptibility gene that regulates T cell activation and tolerance.
In the microwave part of the spectrum, where losses are minimal, metal films regularly patterned (perforated) on the sub-wavelength scale achieve spectral selectivity by balancing the transmission and reflection characteristics of the surface. Here we show for optical frequencies, where joule losses are important, that periodic structuring of a metal film without violation of continuity (i.e. without perforation) is sufficient to achieve substantial modification of reflectivity. By engineering the geometry of the structure imposed on a surface one can dramatically change the perceived color of the metal without employing any form of chemical modification, thin-film coating or diffraction effects. This novel frequency selective effect is underpinned by plasmonic Joule losses in the constituent elements of the patterns (dubbed intaglio and bas relief metamaterials to distinguish indented and raised structures respectively) and is specific to the optical part of the spectrum. It has the advantage of maintaining the integrity of metal surfaces and is well suited to high-throughput fabrication via techniques such as nano-imprint.
The concept of growing nanosize particles on polarized ferroelectric domain areas is known as ferroelectric lithography (FL). Here, a further step of technical development was achieved by combining nanoembossing technique with the FL to realize the selective growth of silver on the polarized areas induced by nanoembossing. The induced rearrangements of domain distributions by embossing in the ferroelectric films have been characterized by piezoresponse force microscopy (PFM). The selective photochemical reduction of silver particles on the embossed nanostructures associated with the underlying domain patterns created by the nanoembossing process has been successfully demonstrated. This nanoembossing induced ferroelectric lithography (NIFL) developed in this work is expected to create an alternative route for nanoscale patterning of metals.
In this work, we apply nano-embossing technique to form a stagger structure in ferroelectric lead zirconate titanate [Pb(Zr0.3, Ti0.7)O3 (PZT)] films and investigate the ferroelectric and electrical characterizations of the embossed and un-embossed regions, respectively, of the same films by using piezoresponse force microscopy (PFM) and Radiant Technologies Precision Material Analyzer. Attributed to the different layer thickness of the patterned ferroelectric thin film, two distinctive coercive voltages have been obtained, thereby, allowing for a single ferroelectric memory cell to contain more than one bit of data.
Hydrogen silsesquioxane (HSQ) is a material with the potential for studying the effect of surface stiffness on stem cell differentiation. Here, the effects of electron beam dose on the topography and the mechanical properties of HSQ obtained with or without trimethylamine (TMA) development are characterised by atomic force microscopy imaging and indentation. A correlation between the surface stiffness (uniform across the sample) and electron beam exposure is observed. Surface roughness of HSQ samples developed in TMA decreases exponentially with increasing electron beam exposure. Surface coating with plasma polymerised allylamine (ppAAm) leads to an overall decrease in stiffness values. However, the increase in surface stiffness with increasing electron beam exposure is still evident. The ppAAm coating is shown to facilitate human mesenchymal stem cell adhesion.
It is well known that photochemical reaction in an aqueous solution can be chosen by selectively patterning the domain structures of ferroelectrics. In this work, we investigate the photochemically induced deposition of Ag particles on ferroelectric lead zirconate titanate [Pb(Zr(x),Ti(1?-?x))O(3)] nanowires fabricated by nanoembossing technology. The photochemical reduction of Ag particles is found to occur preferentially along the embossed nanowires. By imaging domain configurations of the embossed films using the piezoresponse force microscope, the spatially selective deposition of Ag particles can be associated with the underlying ferroelectric domain structures created by the nanoembossing process. The controllable and selective deposition of metal species onto nanoembossed ferroelectric nanostructures without the need for an external electrical field is promising for providing a new route to nanoferroelectric lithography.
Henoch-Schönlein purpura (HSP) is one of the most common causes of systemic vasculitis in children. The incidence of HSP nephritis (HSPN) among HSP patients has been reported to be 15-62%. Even so, what constitutes severe HSPN is controversial. In the study reported here, we retrospectively reviewed the clinical features and prognosis of 101 children with HSPN, ISKDC grade IIIa/IIIb, from January 1992 to November 2008. Patients with isolated hematuria and/or proteinuria?<50 mg/kg/day received triptolide alone, and those with nephrotic range proteinuria received a combination therapy of prednisone and triptolide. Nephrotic syndrome was the most common clinical manifestation (45.5%). There were no significant differences in the clinical features (?(2)?=?2.756, P?=?0.252), the side effects related to treatment (?(2)?=?2.259, P?=?0.894), prognosis between IIIa and IIIb (?(2)?=?3.013, P?=?0.222), or prognosis in grade IIIa patients receiving triptolide alone or triptolide and prednisone (?(2)?=?1.207, P?=?0.272) and grade IIIb patients (?(2)?=?1.158, P?=?0.282). No significant difference in clinical manifestations and long-term prognosis of our HSPN patients with grade IIIa or grade IIIb were found, implying that our patients with International Study and Kidney Disease in Children (ISKDC) grade IIIb were not the most severe cases of HSPN. Our results may also suggest that treatment with steroid may not alter the clinical outcome of such grade IIIa or IIIb patients.
In this work, we have undertaken evaluation of the lithography property of a recently available chemically amplified resist (CAR) resist, UV1116 supplied by Rohm and Haas Company. Systematic study of the EBL property such as sensitivity, contrast, high resolution limit and dense capability, as well as resistance to plasma dry etching has been carried out. In comparison with the performance of UVIII, we conclude that the UV1116 can be a good alternative with better lithography quality.
Cereal starch granules with high-amylose and resistant starch (RS) always show irregular morphology and special crystalline structure, but their formation during grain development is not yet clear. In our previous studies, we had generated a transgenic rice line (TRS) enriched with amylose and RS, which contained semi-compound starch showing a C-type crystalline structure. In this study, the formation of semi-compound C-type starch granule during TRS endosperm development was carefully investigated with light, scanning electron, and transmission electron microscopes and X-ray powder diffraction. The results showed that the TRS starch subgranules, each with a central hilum, were individually initiated in amyloplast and showed an A-type crystal at the early stage of starch granule development, which was similar to that in its wild type. However, with the endosperm development, the amylose content in TRS endosperm starch increased and the B-type starch crystal was deposited in the periphery of subgranules; then, the adjacent subgranules fused together and finally formed a continuous outer layer band surrounding the entire circumference of the starch granule. Accordingly, a mechanistic model for the formation of semi-compound C-type starch granules is proposed.
Skilled placement of peripherally inserted central catheters (PICC) has a profound impact on patient well-being and costs of care. The use of ultrasound-guided methods and prescribed training for cannulation skills are evidence-based practice recommendations. The purpose of this study was to compare two methods of PICC instruction on the acquisition of applied skills.
The evolution of the immune system has provided a multilevel system that interconnects the innate and adaptive immune systems to serve at least three central purposes: the defense from microbial pathogens, the capacity for discrimination of self- from non-self necessary for the prevention of autoimmune disease, and essential effector roles in wound repair and tissue remodeling. In recent studies, we have elucidated an unsuspected role for a class of naturally occurring autoreactive antibodies from the most primitive tier of B lymphocytes, which regulates fundamental functions of the innate immune system. Our findings also throw light onto long unresolved mysteries regarding the origins of the earliest waves of B lymphocyte development.
Although natural Abs (NAbs) are present from birth, little is known about what drives their selection and whether they have housekeeping functions. The prototypic T15-NAb, first identified because of its protective role in infection, is representative of a special type of NAb response that specifically recognizes and forms complexes with apoptotic cells and which promotes cell-corpse engulfment by phagocytes. We now show that this T15-NAb IgM-mediated clearance process is dependent on the recruitment of C1q and mannose-binding lectin, which have known immune modulatory activities that also provide "eat me" signals for enhancing phagocytosis. Further investigation revealed that the addition of T15-NAb significantly suppressed in vitro LPS-induced TNF-alpha and IL-6 secretion by the macrophage-like cell line, RAW264.7, as well as TLR3-, TLR4-, TLR7-, and TLR9-induced maturation and secretion of a range of proinflammatory cytokines and chemokines by bone marrow-derived conventional dendritic cells. Significantly, high doses of this B-1 cell produced NAb also suppressed in vivo TLR-induced proinflammatory responses. Although infusions of apoptotic cells also suppressed such in vivo inflammatory responses and this effect was associated with the induction of high levels of IgM antiapoptotic cell Abs, apoptotic cell treatment was not effective at suppressing such TLR responses in B cell-deficient mice. Moreover, infusions of T15-NAb also efficiently inhibited both collagen-induced arthritis and anti-collagen II Ab-mediated arthritis. These studies identify and characterize a previously unknown regulatory circuit by which a NAb product of innate-like B cells aids homeostasis by control of fundamental inflammatory pathways.
We present the fabrication of 150 nm half-pitch Si grating templates by reactive ion etch (RIE), which are used in nanoimprint lithography (NIL) for high groove density gratings in SU-8 plastic. The etch properties such as the etch rate, profile and etching selectivity of Si over Cr as etch mask were carefully studied. Under the optimum condition Si gratings with 150 nm in linewidth, 480 nm in height and nearly 90 degree in verticality of the sidewall have been achieved. 150 nm half-pitch gratings on SU-8 were then successfully imprinted using the fabricated templates. The diffraction pattern of zeroth and first order from the SU-8 gratings was observed using a 266 nm laser beam. The developed nanofabrication technique in this work is applicable not only for templates but also for other nanostructures in silicon.
Natural Abs, which arise without known immune exposure, have been described that specifically recognize cells dying from apoptosis, but their role in innate immunity remains poorly understood. Herein, we show that the immune response to neoantigenic determinants on apoptotic thymocytes is dominated by Abs to oxidation-associated Ags, phosphorylcholine (PC), a head group that becomes exposed during programmed cell death, and malondialdehyde (MDA), a reactive aldehyde degradation product of polyunsaturated lipids produced following exposure to reactive oxidation species. While natural Abs to apoptotic cells in naive adult mice were dominated by PC and MDA specificities, the amounts of these Abs were substantially boosted by treatment of mice with apoptotic cells. Moreover, the relative amounts of PC and MDA Abs was affected by V(H) gene inheritance. Ab interactions with apoptotic cells also mediated the recruitment of C1q, which enhanced apoptotic cell phagocytosis by immature dendritic cells. Significantly, IgM Abs to both PC and MDA were primary factors in determining the efficiency of serum-dependent apoptotic cell phagocytosis. Hence, we demonstrate a mechanism by which certain natural Abs that recognize neoantigens on apoptotic cells, in naive mice and those induced by immune exposure to apoptotic cells, can enhance the functional capabilities of immature dendritic cells for phagocytic engulfment of apoptotic cells.
Bacterial infection is a key factor in airway inflammation. The present study describes the time-dependent changes in the leukocyte counts and cytokine levels of the bronchoalveolar lavage fluid (BALF) following subacute airway inflammation induced by lipopolysaccharide (LPS), a major component of the outer membranes of Gram-negative bacteria. LPS (200 ?g/rat) or saline was intratracheally administered to rats which were sacrificed 2, 4 or 7 days after LPS treatment. Airway inflammation was evaluated using hematoxylin and eosin staining, cell counts and proinflammatory cytokine levels in the BALF. Rat airways obtained from the LPS group exhibited marked airway wall thickening and infiltration of inflammatory cells compared with the control group, as well as elevated cell counts (neutrophils, macrophages, lymphocytes) and proinflammatory cytokine levels [(tumor necrosis factor (TNF)-?, interleukin (IL)-1?, cytokine-induced neutrophil chemoattractant (CINC)-1)] in the BALF, which peaked on day 2 and subsequently decreased until the experimental endpoint. Notably, IL-1? levels induced by LPS changed in a similar manner to macrophage cell counts, but not neutrophil and lymphocyte counts. Moreover, TNF-? and CINC-1 levels did not decrease as rapidly as neutrophil counts after peaking. These findings suggest that macrophages may play a significant role in maintaining subacute inflammatory responses induced by LPS in rat airways.
Naturally arising IgM antibodies, which recognize neo-determinants on apoptotic cell (AC) membranes, are present from birth and can be further induced by AC challenge. Such naturally arising IgM antibodies can suppress proinflammatory responses to purified agonists for Toll-like receptors (TLRs), as well as block the induction of IgG immune complex-induced in vitro and in vivo pathogenic responses. To investigate the responsible mechanisms, we studied the regulatory effects of IgM anti-AC antibody on responses in bone marrow-derived dendritic cells mediated by a range of different TLRs and found that addition of IgM anti-AC inhibited the activation of the primary MAPKs: ERK1/2, JNK, and particularly p38. This was dependent on the recruitment of either C1q or mannose-binding lectin, which are both early complement factors that tag ACs for innate immune recognition. Strikingly, MAPK inhibition of responses to TLR agonists, and to lupus IgG autoantibody-chromatin immune complexes, was found to correlate with, and had an absolute requirement for, the induction and nuclear localization of MAPK phosphatase-1, a factor known to mediate glucocorticoid suppression of immune responses. Further experiments showed that natural IgM antibodies in serum exhibited the same inhibitory properties. These studies elucidate a novel homeostatic pathway by which natural antibodies, which are products of the adaptive immune system, can directly blunt inflammatory responses by recruitment and coordination of a primitive regulatory pathway of the innate immune system.
Stem and progenitor cells maintain the tissue they reside in for life by regulating the balance between proliferation and differentiation. How this is done is not well understood. Here, we report that the human exosome maintains progenitor cell function. The expression of several subunits of the exosome were found to be enriched in epidermal progenitor cells, which were required to retain proliferative capacity and to prevent premature differentiation. Loss of PM/Scl-75 also known as EXOSC9, a key subunit of the exosome complex, resulted in loss of cells from the progenitor cell compartment, premature differentiation, and loss of epidermal tissue. EXOSC9 promotes self-renewal and prevents premature differentiation by maintaining transcript levels of a transcription factor necessary for epidermal differentiation, GRHL3, at low levels through mRNA degradation. These data demonstrate that control of differentiation specific transcription factors through mRNA degradation is required for progenitor cell maintenance in mammalian tissue.
Glucose-induced reactive oxygen species (ROS) production initiates podocyte apoptosis, which represents a novel early mechanism leading to diabetic nephropathy (DN). Here, we tested the hypothesis that Astragaloside IV(AS-IV) exerts antioxidant and antiapoptotic effects on podocytes under diabetic conditions. Apoptosis, albuminuria, ROS generation, caspase-3 activity and cleavage, as well as Bax and Bcl-2 mRNA and protein expression were measured in vitro and in vivo. Cultured podocytes were exposed to high glucose (HG) with 50, 100 and 200 µg/ml of AS-IV for 24 h. AS-IV significantly attenuated HG-induced podocyte apoptosis and ROS production. This antiapoptotic effect was associated with restoration of Bax and Bcl-2 expression, as well as inhibition of caspase-3 activation and overexpression. In streptozotocin (STZ)-induced diabetic rats, severe hyperglycemia and albuminuria were developed. Increased apoptosis, Bax expression, caspase-3 activity and cleavage while decreased Bcl-2 expression were detected in diabetic rats. However, pretreatment with AS-IV (2.5, 5, 10 mg·kg(-1)·d(-1)) for 14 weeks ameliorated podocyte apoptosis, caspase-3 activation, renal histopathology, podocyte foot process effacement, albuminuria and oxidative stress. Expression of Bax and Bcl-2 mRNA and protein in kidney cortex was partially restored by AS-IV pretreatment. These findings suggested AS-IV, a novel antioxidant, to prevent Glucose-Induced podocyte apoptosis partly through restoring the balance of Bax and Bcl-2 expression and inhibiting caspase-3 activation.
Cyclosporine A (CsA) and tacrolimus (TAC) are often alternative treatment choices for patients with nephrotic syndrome.
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