A TGF-ß-responsive enhancer regulates SRC expression and epithelial-mesenchymal transition-associated cell migration.

The non-receptor tyrosine kinase SRC is overexpressed and/or hyperactivated in various human cancers, and facilitates cancer progression by promoting invasion and metastasis. However, the mechanisms underlying SRC upregulation are poorly understood. In this study, we demonstrate that transforming growth factor-ß (TGF-ß) induces SRC expression at the transcriptional level by activating an intragenic the SRC enhancer. In the human breast epithelial cell line MCF10A, TGF-ß1 stimulation upregulated one of the SRC promotors, the 1A promoter, resulting in increased SRC mRNA and protein levels. Chromatin immunoprecipitation (ChIP)-sequencing analysis revealed that the SMAD complex is recruited to three enhancer regions ∼15 kb upstream and downstream of the SRC promoter, and one of them is capable of activating the SRC promoter in response to TGF-ß. JUN, a member of the activator protein (AP)-1 family, localises to the enhancer and regulates TGF-ß-induced SRC expression. Furthermore, TGF-ß-induced SRC upregulation plays a crucial role in epithelial-mesenchymal transition (EMT)-associated cell migration by activating the SRC-focal adhesion kinase (FAK) circuit. Overall, these results suggest that TGF-ß-induced SRC upregulation promotes cancer cell invasion and metastasis in a subset of human malignancies.

TNFRSF10A downregulation induces retinal pigment epithelium degeneration during the pathogenesis of age-related macular degeneration and central serous chorioretinopathy.

Age-related macular degeneration (AMD) and central serous chorioretinopathy (CSC) are common diseases that can cause vision loss in older and younger populations. These diseases share pathophysiological conditions derived from retinal pigment epithelium (RPE) dysfunction. Tumor necrosis factor receptor superfamily 10A (TNFRSF10A)-LOC389641 with the same lead single-nucleotide polymorphism (SNP) (rs13278062) is the only overlapped susceptibility locus found in both AMD and CSC through genome-wide association studies. This lead SNP has been reported to alter the transcriptional activity of TNFRSF10A. This study aimed to elucidate the function of TNFRSF10A in RPE degeneration using human primary RPE cells and Tnfrsf10 knockout (Tnfrsf10-/-) mice. TNFRSF10A was found to be localized in human RPE. In vitro assays revealed that a T allele of rs13278062, the risk allele for AMD and CSC, downregulated TNFRSF10A transcription in RPE, leading to decreased cell viability and increased apoptosis through protein kinase C-α (PKCA) downregulation. Treatment with phorbol 12-myristate 13-acetate, a PKC activator, rescued the cell viability. Morphological RPE abnormality was found in the retina of Tnfrsf10-/- mice. Our data suggest that downregulation of TNFRSF10A expression inactivates PKCA signaling and causes cellular vulnerability of the RPE, which may contribute to the pathogenesis of AMD and CSC.

Monomorphic T-cell post-transplant lymphoproliferative disorder with features of HHV8-negative primary effusion lymphoma: an autopsy case and review of the literature.

A 67-year-old man underwent renal transplantation in his twenties. He developed refractory pleural effusion, with many large lymphocytes with severe atypia and mitosis in the effusion, indicating malignant lymphoma. He finally died of respiratory failure. An autopsy revealed atypical lymphocytes positive for CD3, CD4, and CD30 and negative for CD8, CD20, PAX5, human herpesvirus (HHV) 8, and Epstein-Barr virus-encoded small RNAs by immunohistochemistry and in situ hybridization. Atypical lymphocytes also had T-cell receptor gene rearrangements Jß2, Jγ2, and Jδ1 and chromosomal aberrations der(8)t(1;8)(q21;p21), add(13)(q12), add(14)(q32), and add(16)(q12-13). A few atypical lymphocytes were present at other sites. We finally diagnosed this case as monomorphic T-cell post-transplant lymphoproliferative disorder with features of HHV8-negative primary effusion lymphoma. A literature review only identified six cases (four HHV8-negative, two HHV8-positive) of effusion lymphoma of T-cell type, including the present case. Interestingly, about half of HHV8-negative and HHV8-positive cases had a history of renal transplantation in their twenties. All cases showed tumor CD30 expression, whereas CD4 and CD8 expressions were inconsistent. These findings indicated that this lymphoma may be associated with post-transplant lymphoproliferative disorder by renal transplantation at a young age, although further cases need to be analyzed.

The liver kinase B1 supports mammary epithelial morphogenesis by inhibiting critical factors that mediate epithelial-mesenchymal transition.

The liver kinase B1 (LKB1) controls cellular metabolism and cell polarity across species. We previously established a mechanism for negative regulation of transforming growth factor ß (TGFß) signaling by LKB1. The impact of this mechanism in the context of epithelial polarity and morphogenesis remains unknown. After demonstrating that human mammary tissue expresses robust LKB1 protein levels, whereas invasive breast cancer exhibits significantly reduced LKB1 levels, we focused on mammary morphogenesis studies in three dimensional (3D) acinar organoids. CRISPR/Cas9-introduced loss-of-function mutations of STK11 (LKB1) led to profound defects in the formation of 3D organoids, resulting in amorphous outgrowth and loss of rotation of young organoids embedded in matrigel. This defect was associated with an enhanced signaling by TGFß, including TGFß auto-induction and induction of transcription factors that mediate epithelial-mesenchymal transition (EMT). Protein marker analysis confirmed a more efficient EMT response to TGFß signaling in LKB1 knockout cells. Accordingly, chemical inhibition of the TGFß type I receptor kinase largely restored the morphogenetic defect of LKB1 knockout cells. Similarly, chemical inhibition of the bone morphogenetic protein pathway or the TANK-binding kinase 1, or genetic silencing of the EMT factor SNAI1, partially restored the LKB1 knockout defect. Thus, LKB1 sustains mammary epithelial morphogenesis by limiting pathways that promote EMT. The observed downregulation of LKB1 expression in breast cancer is therefore predicted to associate with enhanced EMT induced by SNAI1 and TGFß family members.

Transcriptome analysis of Aurantiochytrium limacinum under low salt conditions.

Aurantiochytrium limacinum can accumulate high amounts of omega-3 polyunsaturated fatty acids, especially docosahexaenoic acid (DHA). Although salinity affects the DHA content, its impact on the metabolic pathway responsible for DHA production in A. limacinum is not completely understood. To address this issue, we investigated the transcriptional profile of A. limacinum under hypoosmotic stress. We first cultured A. limacinum under typical and low salinity for RNA sequencing, respectively. Transcriptome analyses revealed that 933 genes exhibited significant changes in expression under hypoosmotic conditions, of which 81.4% were downregulated. Strikingly, A. limacinum downregulated genes related to polyketide synthesis and fatty acid synthase pathways, while upregulating ß-oxidation-related genes. In accordance with this, DHA production significantly decreased under hypoosmotic conditions, while antioxidant-related genes were significantly upregulated. Considering that ß-oxidation of fatty acids generates energy and reactive oxygen species (ROS), our results suggest that A. limacinum utilizes fatty acids for energy to survive under hypoosmotic conditions and detoxifies ROS using antioxidant systems.

An adrenal incidentaloma that had appeared to produce dehydroepiandrosterone-sulfate in excess before immunohistochemical study of the tumor.

Adrenal incidentaloma is a clinically unapparent adrenal mass more than one cm in diameter detected during imaging performed not for adrenal disease. A 34-year-old man was evaluated for AI with a diameter of 3.5 cm in the left adrenal. He was obese with body mass index of 33,9. Blood pressure was 110-120/90 mmHg. The general laboratory tests were unremarkable. An adrenal hormone screening set revealed that ACTH was 6.9 pg/mL, cortisol 14.9 µg/dL, renin activity 0.9 ng/mL/h, aldosterone 79.4 pg/mL, dehydroepiandrosterone-sulfate (DHEA-S) measured on two occasions 5,217 ng/mL and 6,477 ng/mL (gender- and age-adjusted reference values, 1,060-4,640 ng/mL). The levels of metanephrine and normetanephrine were normal. The tumor was thought to produce solely DHEA-S. The excised left adrenal tissue contained a tumor with a diameter of 26 mm and neighboring adrenal tissue. The tumor consisted mostly of acidophil cells without necrosis, capsular or vascular invasion, and mitosis. Immunohistochemical study revealed followings: the cells of the tumors were stained positive for 3ß-hydroxysteroid dehydrogenase, and 17α-hydroxylase, and 11ß-hydroxylase, weakly positive for DHEA sulphotransferase, and negative for aldosterone synthetase. The atrophy of neighboring tissue was presumably caused by excess cortisol production. Four months after surgery, the cortisol level was 11.2 µg/dL and DHEA-S level 1,462 ng/mL. The tumor is considered to be a cortisol-producing adenoma with modestly excessive DHEA-S production rather than isolated DHEA-S-producing adenoma. Immunohistochemical study of steroidogenic enzymes is a valuable addition to blood hormone measurement to clarify steroid production profile.

Transcriptional responses of Aurantiochytrium limacinum under light conditions.

AIMS: Astaxanthin-producing protist Aurantiochytrium limacinum can accumulate higher amounts of astaxanthin under light conditions; however, little is known about the impact of light exposure on its metabolism. Here, we investigated the transcriptional profile of A. limacinum under light conditions. METHODS AND RESULTS: Transcriptomic analyses revealed that 962 genes of A. limacinum showed a significant change in expression under light conditions, most of which (94.5%) were downregulated. Furthermore, gene ontology enrichment analysis indicated that A. limacinum mainly downregulated genes associated with cell motility, proliferation and gene expression processes, whose activities depend on ATP as an energy source. Additionally, the quantification of carotenoid and its transcripts suggested that ß-carotene and astaxanthin biosynthesis pathways were rate-limiting and tightly regulated steps, respectively. In comparison, these processes were enhanced under light conditions. CONCLUSIONS: Considering that astaxanthin accumulation was highly correlated with reactive oxygen species (ROS) levels in microalgae, our results suggest that A. limacinum reduces ATP consumption to decrease the occurrence of ROS in mitochondria while accumulating astaxanthin to prevent ROS damage. SIGNIFICANCE AND IMPACT OF STUDY: This study provides novel insights into the impact of light exposure on A. limacinum metabolism, thereby facilitating a complete understanding of this protist for efficient astaxanthin production.

Porous Self-Assembled Molecular Networks as Templates for Chiral-Position-Controlled Chemical Functionalization of Graphitic Surfaces.

Controlled covalent functionalization of graphitic surfaces with molecular scale precision is crucial for tailored modulation of the chemical and physical properties of carbon materials. We herein present that porous self-assembled molecular networks (SAMNs) act as nanometer scale template for the covalent electrochemical functionalization of graphite using an aryldiazonium salt. Hexagonally aligned achiral grafted species with lateral periodicity of 2.3, 2.7, and 3.0 nm were achieved utilizing SAMNs having different pore-to-pore distances. The unit cell vectors of the grafted pattern match those of the SAMN. After the covalent grafting, the template SAMNs can be removed by simple washing with a common organic solvent. We briefly discuss the mechanism of the observed pattern transfer. The unit cell vectors of the grafted pattern align along nonsymmetry axes of graphite, leading to mirror image grafted domains, in accordance with the domain-specific chirality of the template. In the case in which a homochiral building block is used for SAMN formation, one of the 2D mirror image grafted patterns is canceled. This is the first example of a nearly crystalline one-sided or supratopic covalent chemical functionalization. In addition, the positional control imposed by the SAMN renders the functionalized surface (homo)chiral reaching a novel level of control for the functionalization of carbon surfaces, including surface-supported graphene.

Steric and Electronic Effects of Electrochemically Generated Aryl Radicals on Grafting of the Graphite Surface.

Grafting of aryl radicals generated by electrochemical reduction of aryldiazonium salts has been extensively studied on various surfaces. However, there exists two unclear aspects; the first one is the generality of the blocking ability of simple functional groups toward multilayer growth, and the second one is the electronic impact of substituent groups of aryl radicals on grafting efficiency. To address these aspects, we have studied the electrochemical functionalization of graphite using aryldiazonium salts having electron-donating or electron-withdrawing groups at the 3,4,5-positions. Atomic force microscopy investigation of the functionalized surfaces revealed the formation of monolayers for all aryldiazonium salts, and thus, nitro, carboxy, ester, methyl, and methoxy groups at the 3,4,5-positions of the benzene ring suppress polyaryl growth. The degree of grafting estimated by scanning tunneling microscopy imaging and Raman spectroscopy of the functionalized surfaces depends on the electronic state of the aryl radicals, in which the radicals with electron-donating groups show a high degree of functionalization, whereas those with electron-withdrawing groups exhibit a low degree of functionalization. We discuss several possibilities that affect grafting density. Though there are several factors, we hypothesize that one factor to explain the observed reactivity trend is the electronic property of the aryl radicals, namely, the relative position of the singly occupied molecular orbital energy levels of the aryl radicals with respect to the graphite Fermi energy level.

An autopsy case of globular glial tauopathy presenting with clinical features of motor neuron disease with dementia and iron deposition in the motor cortex.

Globular glial tauopathy (GGT) is a 4-repeat (4R) tauopathy in which 4R tau accumulates to form globular glial inclusions (GGIs), predominantly in oligodendroglia. To date, little has been reported on iron deposits in patients with GGT. We report a case of GGT with iron deposits in a 78-year-old woman presenting with an 8-year history of slowly progressing limb weakness and cognitive decline. Susceptibility-weighted imaging revealed a low signal intensity in the right precentral gyrus, suggesting iron deposition. A clinical diagnosis of motor neuron disease with dementia was made 4 years after onset. At autopsy, gross pathological findings showed atrophy of the frontal and temporal lobes. A localized area of the precentral gyrus corresponding to the most severely affected limb showed the strongest atrophy, macroscopically, and displayed 4R tau-immunoreactive GGIs and microscopically many ferritin-immunoreactive neurons. We diagnosed this patient as having GGT. This is the first GGT case with iron deposition confirmed both radiologically and pathologically.

Visual Outcomes Based on Early Response to Anti-Vascular Endothelial Growth Factor Treatment for Diabetic Macular Edema.

OBJECTIVE: To examine the relationship between early response to anti-vascular endothelial growth factor (VEGF) treatment and visual prognosis. METHODS: We retrospectively separated 20 patients with persistent diabetic macular edema (DME) into two responder status groups based on the reduction of central macular thickness (CMT) from baseline to month 3: a delayed responder group (DRG) (≤25% CMT reduction, n = 11) and an immediate responder group (IRG) (>25% CMT reduction, n = 14). We also separated the patients into two responder status groups based on the logarithm of the minimum angle of resolution (logMAR) best-corrected visual acuity (BCVA): a visual nonimprovement group (VNIG) (≥0 logMAR BCVA improvement, n = 11) and a vi sual improvement group (VIG) (<0 logMAR BCVA improvement, n = 14). Finally, we assessed the correlations between logMAR BCVA changes from baseline to month 3 (ΔBCVAM3) and those from baseline to month 12 (ΔBCVAM12). RESULTS: At month 12, BCVA was significantly more improved in the VIG than the VNIG (p < 0.005), but was not significantly different between the DRG and the IRG (p = 0.75). The Pearson correlation coefficient showed a significant relationship between ΔBCVAM3 and ΔBCVAM12 (r = 0.60, p < 0.005). CONCLUSIONS: BCVA showed significantly greater improvement in the VIG than in the VNIG. ΔBCVAM3 may predict the visual outcome at month 12 in DME patients treated with anti-VEGF drugs.

[Spontaneous Rupture of the Ascending Aorta;Report of a Case].

A 66-year-old woman was transferred to our hospital on an emergency basis complaining of a sudden onset of chest discomfort. An emergency contrasted computed tomography showed a ruptured the ascending aorta near the right pulmonary artery with a hematoma and notch in the aortic wall. An emergency operation of the graft replacement was performed via standard median sternotomy under the cardiac arrest with aortic clamp. Upon the operation, there was a 1.5 cm rupture site in the central portion of the ascending aorta. There was no specific evidence of aortic aneurysm or dissection, therefore a spontaneous rupture of the ascending aorta was diagnosis. The pathological finding was normal without pathological changes of cystic medial necrosis or atheroma. The postoperative course was uneventful and he was discharged 14 days after surgery.

Comparación de la efectividad de ranibizumab intravítreo para el tratamiento del edema macular diabético en ojos vitrectomizados y no vitrectomizados.

Objetivo: Comparar la efectividad de ranibizumab intravítreo (RIV) para el tratamiento del edema macular diabético (EMD) en ojos con y sin vitrectomía previa. Procedimientos: Evaluamos de manera prospectiva la mejor agudeza visual corregida (MAVC) y el grosor macular central (GMC) tras el tratamiento con RIV durante 6 meses. Resultados: No se observaron diferencias significativas en la MAVC o GMC inicial en ninguno de los dos grupos. En el grupo no vitrectomizado (n = 15), los cambios medios en la MAVC y GMC hasta el sexto mes de tratamiento con respecto al valor inicial resultaron significativos (p < 0,01). En el grupo vitrectomizado (n = 10), se observó una mejora más lenta, y la mejora media en la MAVC no resultó significativa (p = 0,5), aunque la media en la disminución del GMC sí que lo fue (p < 0,05). No se observaron diferencias significativas en los cambios medios en la MAVC y el GMC entre ambos grupos a los 6 meses del tratamiento. Conclusiones: La diferencia en la efectividad de RIV entre ambos grupos no resultó significativa. Ranibizumab intravítreo puede ser una opción de tratamiento incluso en pacientes vitrectomizados con EMD.

A Case of Diabetic Macular Edema with Improvement in Severity of Diabetic Retinopathy following Frequent Anti-Vascular Endothelial Growth Factor Treatment.

Background: Intravitreal anti-vascular endothelial growth factor (VEGF) drug injections are the gold standard for the treatment of diabetic macular edema (DME). We report a case of DME in which frequent anti-VEGF treatment resulted in improvement of diabetic retinopathy. Case: A 73-year old woman with DME who had previously undergone bilateral posterior subtenon injections of triamcinolone acetonide and vitrectomy OD presented at Kyushu University Hospital. Best corrected visual acuity was 0.1 OD and 0.15 OS. The central macular thickness was 1570 µm OD and 578 µm OS. We performed focal macular laser photocoagulation OU but the DME was not resolved. Subsequent intravitreal anti-VEGF drug injections(approximately 20 times/2 years)resulted in an improvement of the best corrected visual acuity (0.3 OD, 0.6 OS) and CMT (1570 µm OD, 578 µm OS). There was an improvement of 2 steps in the Diabetic Retinopathy Severity Scale (DRSS) score OU. Conclusion: Frequent anti-VEGF treatment can improve the severity of diabetic retinopathy.

Tenascin-C secreted by transdifferentiated retinal pigment epithelial cells promotes choroidal neovascularization via integrin αV.

Tenascin-C is expressed in choroidal neovascular (CNV) membranes in eyes with age-related macular degeneration (AMD). However, its role in the pathogenesis of CNV remains to be elucidated. Here we investigated the role of tenascin-C in CNV formation. In immunofluorescence analyses, tenascin-C co-stained with α-SMA, pan-cytokeratin, CD31, CD34, and integrin αV in the CNV membranes of patients with AMD and a mouse model of laser-induced CNV. A marked increase in the expression of tenascin-C mRNA and protein was observed 3 days after laser photocoagulation in the mouse CNV model. Tenascin-C was also shown to promote proliferation and inhibit adhesion of human retinal pigment epithelial (hRPE) cells in vitro. Moreover, tenascin-C promoted proliferation, adhesion, migration, and tube formation in human microvascular endothelial cells (HMVECs); these functions were, however, blocked by cilengitide, an integrin αV inhibitor. Exposure to TGF-ß2 increased tenascin-C expression in hRPE cells. Conditioned media harvested from TGF-ß2-treated hRPE cell cultures enhanced HMVEC proliferation and tube formation, which were inhibited by pretreatment with tenascin-C siRNA. The CNV volume was significantly reduced in tenascin-C knockout mice and tenascin-C siRNA-injected mice. These findings suggest that tenascin-C is secreted by transdifferentiated RPE cells and promotes the development of CNV via integrin αV in a paracrine manner. Therefore, tenascin-C could be a potential therapeutic target for the inhibition of CNV development associated with AMD.

Comparison of the Effectiveness of Intravitreal Ranibizumab for Diabetic Macular Edema in Vitrectomized and Nonvitrectomized Eyes.

PURPOSE: To compare the effectiveness of intravitreal ranibizumab (IVR) for diabetic macular edema (DME) between eyes with and without previous vitrectomy. PROCEDURES: We prospectively assessed the best-corrected visual acuity (BCVA) and central macular thickness (CMT) after IVR for 6 months. RESULTS: There were no significant differences in the baseline BCVA and CMT between both groups. In the nonvitrectomized group (n = 15), the mean changes of BCVA and CMT from baseline to month 6 were significant (p < 0.01). In the vitrectomized group (n = 10), the improvement appeared to be slower, and the mean BCVA improvement was not significant (p = 0.5), although the mean CMT decrease was significant (p < 0.05). There were no significant differences in the mean changes of BCVA and CMT between both groups at 6 months. CONCLUSIONS: The difference in the effectiveness of IVR between both groups was not significant. IVR can be a treatment option even for vitrectomized DME eyes.

[Familial Hemophagocytic Lymphohistiocytosis, That was Considered as Progressive Encephalitis before Sibling was Diagnosed].

Familial hemophagocytic lymphohistiocytosis (FHL) is an autosomal recessive disorder that is clinically characterized by fever, hepatosplenomegaly, cytopenia and sometimes vague or dramatic central nervous system (CNS) dysfunction. FHL affecting the CNS imitates several neurologic disorders and may be misdiagnosed, in particular when family history is unknown. We report an autopsy case of FHL that was firstly considered as progressive encephalitis. FHL was suspected after sibling had been affected by hemophagocytosis and the same CNS symptoms. Histopathologically, lymphocytes and macrophages infiltrated into the meninges, perivascular space, and parenchyma of the brain. Those lymphocytes were positive for CD3, CD8, GranzymeB, and negative for CD4, perforin. FHL must be included in the differential diagnostic considerations in children with progressive encephalitis.

In vitro assembly properties of human type I and II hair keratins.

Multiple type I and II hair keratins are expressed in hair-forming cells but the role of each protein in hair fiber formation remains obscure. In this study, recombinant proteins of human type I hair keratins (K35, K36 and K38) and type II hair keratins (K81 and K85) were prepared using bacterial expression systems. The heterotypic subunit interactions between the type I and II hair keratins were characterized using two-dimensional gel electrophoresis and surface plasmon resonance (SPR). Gel electrophoresis showed that the heterotypic complex-forming urea concentrations differ depending on the combination of keratins. K35-K85 and K36-K81 formed relatively stable heterotypic complexes. SPR revealed that soluble K35 bound to immobilized K85 with a higher affinity than to immobilized K81. The in vitro intermediate filament (IF) assembly of the hair keratins was explored by negative-staining electron microscopy. While K35-K81, K36-K81 and K35-K36-K81 formed IFs, K35-K85 afforded tight bundles of short IFs and large paracrystalline assemblies, and K36-K85 formed IF tangles. K85 promotes lateral association rather than elongation of short IFs. The in vitro assembly properties of hair keratins depended on the combination of type I and II hair keratins. Our data suggest the functional significance of K35-K85 and K36-K81 with distinct assembly properties in the formation of macrofibrils.

A computational and experimental approach reveals that the 5'-proximal region of the 5'-UTR has a Cis-regulatory signature responsible for heat stress-regulated mRNA translation in Arabidopsis.

Translation of specific plant mRNAs is differentially regulated under certain abiotic stress conditions such as heat, oxygen deprivation and dehydration. The majority of transcripts exhibit varying degrees of translational repression, whereas a subset of transcripts escape such repression and remain actively translated. The underlying mechanisms that mediate this control, and in particular the identities of the regulatory RNA elements involved, remain poorly understood. Using a combined computational and experimental approach, we identified a novel cis-regulatory element in the 5'-untranslated region (5'-UTR) that affects differential translation in response to heat stress (HS) in Arabidopsis thaliana. First, we selected a set of genes with distinct translational responses to HS, based on our previously reported genome-wide data regarding changes in polysome loading induced by HS in A. thaliana cultured cells. We evaluated the 5'-UTRs of these messages for their ability to mediate expression, when fused to reporter mRNAs, in protoplasts under HS. The data from the reporter assay and the nucleotide sequences of the 5'-UTRs tested were used to define regulatory elements in the 5'-UTRs, with the help of a partial least square (PLS) regression model. The computational analysis using PLS and subsequent experimental characterization of a series of 5'-UTR mutants provided evidence that the 5'-proximal sequence of the 5'-UTR is a primary and position-dependent determinant of 5'-UTR-mediated differential translation in response to HS. Finally, we discuss the possible mechanism underlying HS regulation of differential mRNA translation.