mitoTALEN Eliminates Mutant mtDNA Genomes in Neurons

Mutations in the mitochondrial DNA (mtDNA) commonly cause severe encephalopathies. Because most of these mtDNA alterations are heteroplasmic, we used a mitochondrial-targeted TALEN (mitoTALEN) to specifically eliminate the mutant mtDNA in the CNS of a mouse model harboring a heteroplasmic mutation in the mitochondrial tRNA alanine gene (m.5024C>T). Delivery to neurons was achieved by using AAV-PHP.eB and neuronal expression was obtained by using a neuronal-specific synapsin promoter. We found that most CNS regions were effectively transduced and showed a significant reduction in mutant mtDNA. This reduction was accompanied by an increase in mitochondrial tRNA alanine level, which is drastically reduced by the mutation. These results showed, for the first time, that mitochondrial-targeted gene editing can be effective in reducing CNS mutant mtDNA in vivo, paving the way for clinical trials in patients with mitochondrial encephalopathies

Detection of interstellar oxidaniumyl: abundant H2O+ towards the star-forming regions DR21, Sgr B2, and NGC6334

We identify a prominent absorption feature at 1115 GHz, detected in first HIFI spectra towards high-mass star-forming regions, and interpret its astrophysical origin. The characteristic hyperfine pattern of the H2O+ ground-state rotational transition, and the lack of other known low-energy transitions in this frequency range, identifies the feature as H2O+ absorption against the dust continuum background and allows us to derive the velocity profile of the absorbing gas. By comparing this velocity profile with velocity profiles of other tracers in the DR21 star-forming region, we constrain the frequency of the transition and the conditions for its formation. In DR21, the velocity distribution of H2O+ matches that of the [CII] line at 158\mu\m and of OH cm-wave absorption, both stemming from the hot and dense clump surfaces facing the HII-region and dynamically affected by the blister outflow. Diffuse foreground gas dominates the absorption towards Sgr B2. The integrated intensity of the absorption line allows us to derive lower limits to the H2O+ column density of 7.2e12 cm^-2 in NGC 6334, 2.3e13 cm^-2 in DR21, and 1.1e15 cm^-2 in Sgr B2.Comment: Accepted for publication in A&

Downregulation of TGF-beta receptor types II and III in oral squamous cell carcinoma and oral carcinoma-associated fibroblasts

<p>Abstract</p> <p>Background</p> <p>The purpose of this study was to assess the expression levels for TβRI, TβRII, and TβRIII in epithelial layers of oral premalignant lesions (oral leukoplakia, OLK) and oral squamous cell carcinoma (OSCC), as well as in oral carcinoma-associated fibroblasts (CAFs), with the final goal of exploring the roles of various types of TβRs in carcinogenesis of oral mucosa.</p> <p>Methods</p> <p>Normal oral tissues, OLK, and OSCC were obtained from 138 previously untreated patients. Seven primary human oral CAF lines and six primary normal fibroblast (NF) lines were established successfully via cell culture. The three receptors were detected using immunohistochemical (IHC), quantitative RT-PCR, and Western blot approaches.</p> <p>Results</p> <p>IHC signals for TβRII and TβRIII in the epithelial layer decreased in tissue samples with increasing disease aggressiveness (P < 0.05); no expression differences were observed for TβRI, in OLK and OSCC (P > 0.05); and TβRII and TβRIII were significantly downregulated in CAFs compared with NFs, at the mRNA and protein levels (P < 0.05). Exogenous expression of TGF-β1 led to a remarkable decrease in the expression of TβRII and TβRIII in CAFs (P < 0.05).</p> <p>Conclusion</p> <p>This study provides the first evidence that the loss of TβRII and TβRIII expression in oral epithelium and stroma is a common event in OSCC. The restoration of the expression of TβRII and TβRIII in oral cancerous tissues may represent a novel strategy for the treatment of oral carcinoma.</p

Autoantibodies to muscarinic acetylcholine receptors found in patients with primary biliary cirrhosis

<p>Abstract</p> <p>Background</p> <p>Autoantibodies to the human muscarinic acetylcholine receptor of the M3 type (hmAchR M3) have been suggested to play an etiopathogenic role in Sjögren's syndrome. Primary biliary cirrhosis (PBC) often is associated with this syndrome. Therefore, we studied the co-presence of hmAchR M3 autoantibodies in patients with PBC.</p> <p>Methods</p> <p>Frequency of hmAchR M3 autoantibodies was assessed by Western blotting analysis as well as by an ELISA using a 25-mer peptide of the 2^ndextracellular loop of hmAchR M3. Co-localization of hmAchR M3/PBC-specific autoantibodies was studied by confocal laser scanning microscopy. Finally, sera from patients with PBC as well as from healthy controls were tested.</p> <p>Results</p> <p>Western blotting analysis as well as results from ELISA testing revealed a significantly enhanced IgG reactivity in PBC patients in contrast to healthy controls. Co-localization of autoantibodies with the hmAchR M3 receptor-specific autoantibodies was observed in 10 out of 12 PBC-patients but none of the 5 healthy controls. Antibodies of the IgM type were not found to be affected.</p> <p>Conclusions</p> <p>For the first time, our data demonstrate the presence of autoantibodies to the hmAchR M3 in PBC patients. These findings might contribute to the understanding of the pathogenesis of this disease. Further studies have to focus on the functionality of hmAchR M3 autoantibodies in PBC patients.</p

TGF-beta receptor 2 downregulation in tumour-associated stroma worsens prognosis and high-grade tumours show more tumour-associated macrophages and lower TGF-beta1 expression in colon carcinoma: a retrospective study

<p>Abstract</p> <p>Background</p> <p>Histological phenotype and clinical behaviour of malignant tumours are not only dependent on alterations in the epithelial cell compartment, but are affected by their interaction with inflammatory cells and tumour-associated stroma. Studies in animal models have shown influence of tumour-associated macrophages (TAM) on histological grade of differentiation in colon carcinoma. Disruption of transforming growth factor beta (TGF-beta) signalling in tumour cells is related to more aggressive clinical behaviour. Expression data of components of this pathway in tumour-associated stroma is limited.</p> <p>Methods</p> <p>Tissue micro arrays of 310 colon carcinomas from curatively resected patients in UICC stage II and III were established. In a first step we quantified amount of CD68 positive TAMs and expression of components of TGF-beta signalling (TGF-beta1, TGF-beta receptors type 1 and 2, Smad 3 and 4) in tumour and associated stroma. Further we analyzed correlation to histological and clinical parameters (histological grade of differentiation (low-grade (i.e. grade 1 and 2) vs. high-grade (i.e. grade 3 and 4)), lymph node metastasis, distant metastasis, 5 year cancer related survival) using Chi-square or Fisher's exact test, when appropriate, to compare frequencies, Kaplan-Meier method to calculate 5-year rates of distant metastases and cancer-related survival and log rank test to compare the rates of distant metastases and survival. To identify independent prognostic factors Cox regression analysis including lymph node status and grading was performed.</p> <p>Results</p> <p>High-grade tumours and those with lymph node metastases showed higher rates of TAMs and lower expression of TGF-beta1. Loss of nuclear Smad4 expression in tumor was associated with presence of lymph node metastasis, but no influence on prognosis could be demonstrated. Decrease of both TGF-beta receptors in tumour-associated stroma was associated with increased lymph node metastasis and shorter survival. Stromal TGF-beta receptor 2 expression was an independent prognostic factor for cancer related survival.</p> <p>Conclusion</p> <p>Histological phenotype and clinical behaviour of colon cancer is not only influenced by mutational incidents in tumour cells but also affected by interaction of tumour tissue with inflammatory cells like macrophages and associated stroma and TGF-beta signalling is one important part of this crosstalk. Further studies are needed to elucidate the exact mechanisms.</p

TGFBR2 and BAX Mononucleotide Tract Mutations, Microsatellite Instability, and Prognosis in 1072 Colorectal Cancers

Mononucleotide tracts in the coding regions of the TGFBR2 and BAX genes are commonly mutated in microsatellite instability-high (MSI-high) colon cancers. The receptor TGFBR2 plays an important role in the TGFB1 (transforming growth factor-β, TGF-β) signaling pathway, and BAX plays a key role in apoptosis. However, a role of TGFBR2 or BAX mononucleotide mutation in colorectal cancer as a prognostic biomarker remains uncertain.We utilized a database of 1072 rectal and colon cancers in two prospective cohort studies (the Nurses' Health Study and the Health Professionals Follow-up Study). Cox proportional hazards model was used to compute mortality hazard ratio (HR), adjusted for clinical, pathological and molecular features including the CpG island methylator phenotype (CIMP), LINE-1 methylation, and KRAS, BRAF and PIK3CA mutations. MSI-high was observed in 15% (162/1072) of all colorectal cancers. TGFBR2 and BAX mononucleotide mutations were detected in 74% (117/159) and 30% (48/158) of MSI-high tumors, respectively. In Kaplan-Meier analysis as well as univariate and multivariate Cox regression analyses, compared to microsatellite stable (MSS)/MSI-low cases, MSI-high cases were associated with superior colorectal cancer-specific survival [adjusted HR, 0.34; 95% confidence interval (CI), 0.20-0.57] regardless of TGFBR2 or BAX mutation status. Among MSI-high tumors, TGFBR2 mononucleotide mutation was associated with CIMP-high independent of other variables [multivariate odds ratio, 3.57; 95% CI, 1.66-7.66; p = 0.0011].TGFBR2 or BAX mononucleotide mutations are not associated with the patient survival outcome in MSI-high colorectal cancer. Our data do not support those mutations as prognostic biomarkers (beyond MSI) in colorectal carcinoma

Detection of interstellar oxidaniumyl: Abundant H2O+ towards the star-forming regions DR21, Sgr B2, and NGC6334

Aims. We identify a prominent absorption feature at 1115 GHz, detected in first HIFI spectra towards high-mass star-forming regions, and interpret its astrophysical origin. Methods. The characteristic hyperfine pattern of the H2O+ ground-state rotational transition, and the lack of other known low-energy transitions in this frequency range, identifies the feature as H2O+ absorption against the dust continuum background and allows us to derive the velocity profile of the absorbing gas. By comparing this velocity profile with velocity profiles of other tracers in the DR21 star-forming region, we constrain the frequency of the transition and the conditions for its formation. Results. In DR21, the velocity distribution of H2O+ matches that of the [Cii] line at 158 μm and of OH cm-wave absorption, both stemming from the hot and dense clump surfaces facing the Hii-region and dynamically affected by the blister outflow. Diffuse foreground gas dominates the absorption towards Sgr B2. The integrated intensity of the absorption line allows us to derive lower limits to the H2O+ column density of 7.2 × 1012 cm−2 in NGC 6334, 2.3 × 1013 cm−2 in DR21, and 1.1 × 1015 cm−2 in Sgr B2

Tumour–stroma interactions in colorectal cancer: converging on β-catenin activation and cancer stemness

Sporadic cases of colorectal cancer are primarily initiated by gene mutations in members of the canonical Wnt pathway, ultimately resulting in β-catenin stabilisation. Nevertheless, cells displaying nuclear β-catenin accumulation are nonrandomly distributed throughout the tumour mass and preferentially localise along the invasive front where parenchymal cells are in direct contact with the stromal microenvironment. Here, we discuss the putative role played by stromal cell types in regulating β-catenin intracellular accumulation in a paracrine fashion. As such, the tumour microenvironment is likely to maintain the cancer stem cell phenotype in a subset of cells, thus mediating invasion and metastasis