MTO1-deficient mouse model mirrors the human phenotype showing complex I defect and cardiomyopathy.

Recently, mutations in the mitochondrial translation optimization factor 1 gene (MTO1) were identified as causative in children with hypertrophic cardiomyopathy, lactic acidosis and respiratory chain defect. Here, we describe an MTO1-deficient mouse model generated by gene trap mutagenesis that mirrors the human phenotype remarkably well. As in patients, the most prominent signs and symptoms were cardiovascular and included bradycardia and cardiomyopathy. In addition, the mutant mice showed a marked worsening of arrhythmias during induction and reversal of anaesthesia. The detailed morphological and biochemical workup of murine hearts indicated that the myocardial damage was due to complex I deficiency and mitochondrial dysfunction. In contrast, neurological examination was largely normal in Mto1-deficient mice. A translational consequence of this mouse model may be to caution against anaesthesia-related cardiac arrhythmias which may be fatal in patients.

Abnormal brain iron metabolism in Irp2 deficient mice is associated with mild neurological and behavioral impairments.

Iron Regulatory Protein 2 (Irp2, Ireb2) is a central regulator of cellular iron homeostasis in vertebrates. Two global knockout mouse models have been generated to explore the role of Irp2 in regulating iron metabolism. While both mouse models show that loss of Irp2 results in microcytic anemia and altered body iron distribution, discrepant results have drawn into question the role of Irp2 in regulating brain iron metabolism. One model shows that aged Irp2 deficient mice develop adult-onset progressive neurodegeneration that is associated with axonal degeneration and loss of Purkinje cells in the central nervous system. These mice show iron deposition in white matter tracts and oligodendrocyte soma throughout the brain. A contrasting model of global Irp2 deficiency shows no overt or pathological signs of neurodegeneration or brain iron accumulation, and display only mild motor coordination and balance deficits when challenged by specific tests. Explanations for conflicting findings in the severity of the clinical phenotype, brain iron accumulation and neuronal degeneration remain unclear. Here, we describe an additional mouse model of global Irp2 deficiency. Our aged Irp2-/- mice show marked iron deposition in white matter and in oligodendrocytes while iron content is significantly reduced in neurons. Ferritin and transferrin receptor 1 (TfR1, Tfrc), expression are increased and decreased, respectively, in the brain from Irp2-/- mice. These mice show impairments in locomotion, exploration, motor coordination/balance and nociception when assessed by neurological and behavioral tests, but lack overt signs of neurodegenerative disease. Ultrastructural studies of specific brain regions show no evidence of neurodegeneration. Our data suggest that Irp2 deficiency dysregulates brain iron metabolism causing cellular dysfunction that ultimately leads to mild neurological, behavioral and nociceptive impairments.

Simultaneous detection of ricin and abrin DNA by real-time PCR (qPCR).

Ricin and abrin are two of the most potent plant toxins known and may be easily obtained in high yield from the seeds using rather simple technology. As a result, both toxins are potent and available toxins for criminal or terrorist acts. However, as the production of highly purified ricin or abrin requires sophisticated equipment and knowledge, it may be more likely that crude extracts would be used by non-governmental perpetrators. Remaining plant-specific nucleic acids in these extracts allow the application of a real-time PCR (qPCR) assay for the detection and identification of abrin or ricin genomic material. Therefore, we have developed a duplex real-time PCR assays for simultaneous detection of ricin and abrin DNA based on the OmniMix HS bead PCR reagent mixture. Novel primers and hybridization probes were designed for detection on a SmartCycler instrument by using 5'-nuclease technology. The assay was thoroughly optimized and validated in terms of analytical sensitivity. Evaluation of the assay sensitivity by probit analysis demonstrated a 95% probability of detection at 3 genomes per reaction for ricin DNA and 1.2 genomes per reaction for abrin DNA. The suitability of the assays was exemplified by detection of ricin and abrin contaminations in a food matrix.

Isolation, preliminary characterization, and full-genome analyses of tick-borne encephalitis virus from Mongolia.

Tick-borne encephalitis virus (TBEV) causes one of the most important inflammatory diseases of the central nervous system, namely severe encephalitis in Europe and Asia. Since the 1980s tick-borne encephalitis is known in Mongolia with increasing numbers of human cases reported during the last years. So far, however, data on TBEV strains are still sparse. We herein report the isolation of a TBEV strain from Ixodes persulcatus ticks collected in Mongolia in 2010. Phylogenetic analysis of the E-gene classified this isolate as Siberian subtype of TBEV. The Mongolian TBEV strain showed differences in virus titers, plaque sizes, and growth properties in two human neuronal cell-lines. In addition, the 10,242 nucleotide long open-reading frame and the corresponding polyprotein sequence were revealed. The isolate grouped in the genetic subclade of the Siberian subtype. The strain Zausaev (AF527415) and Vasilchenko (AF069066) had 97 and 94 % identity on the nucleotide level. In summary, we herein describe first detailed data regarding TBEV from Mongolia. Further investigations of TBEV in Mongolia and adjacent areas are needed to understand the intricate dispersal of this virus.

Monocarboxylate transporter 8 deficiency: altered thyroid morphology and persistent high triiodothyronine/thyroxine ratio after thyroidectomy.

CONTEXT: Thyroid hormone transport across the plasma membrane depends on transmembrane transport proteins, including monocarboxylate transporter 8 (MCT8). Mutations in MCT8 (or SLC16A2) lead to a severe form of X-linked psychomotor retardation, which is characterised by elevated plasma triiodothyronine (T(3)) and low/normal thyroxine (T(4)). MCT8 contributes to hormone release from the thyroid gland. OBJECTIVE: To characterise the potential impact of MCT8-deficiency on thyroid morphology in a patient and in Mct8-deficient mice. DESIGN: Thyroid morphology in a patient carrying the A224V mutation was followed by ultrasound imaging for over 10 years. After thyroidectomy, a histopathological analysis was carried out. The findings were compared with histological analyses of mouse thyroids from the Mct8(-/y) model. RESULTS: We show that an inactivating mutation in MCT8 leads to a unique, progressive thyroid follicular pathology in a patient. After thyroidectomy, histological analysis revealed gross morphological changes, including several hyperplastic nodules, microfollicular areas with stromal fibrosis and a small focus of microfollicular structures with nuclear features reminiscent of papillary thyroid carcinoma (PTC). These findings are supported by an Mct8-null mouse model in which we found massive papillary hyperplasia in 6- to 12-month-old mice and nuclear features consistent with PTC in almost 2-year-old animals. After complete thyroidectomy and substitution with levothyroxine (l-T(4)), the preoperative, inadequately low T(4) and free T(4) remained, while increasing the l-T(4) dosage led to T(3) serum concentrations above the normal range. CONCLUSIONS: Our results implicate peripheral deiodination in the peculiar hormonal constellation of MCT8-deficient patients. Other MCT8-deficient patients should be closely monitored for potential thyroid abnormalities.

A novel N-ethyl-N-nitrosourea-induced mutation in phospholipase Cγ2 causes inflammatory arthritis, metabolic defects, and male infertility in vitro in a murine model.

OBJECTIVE: It is difficult to identify a single causative factor for inflammatory arthritis because of the multifactorial nature of the disease. This study was undertaken to dissect the molecular complexity of systemic inflammatory disease, utilizing a combined approach of mutagenesis and systematic phenotype screening in a murine model. METHODS: In a large-scale N-ethyl-N-nitrosourea mutagenesis project, the Ali14 mutant mouse strain was established because of dominant inheritance of spontaneous swelling and inflammation of the hind paws. Genetic mapping and subsequent candidate gene sequencing were conducted to find the causative gene, and systematic phenotyping of Ali14/+ mice was performed in the German Mouse Clinic. RESULTS: A novel missense mutation in the phospholipase Cγ2 gene (Plcg2) was identified in Ali14/+ mice. Because of the hyperreactive external entry of calcium observed in cultured B cells and other in vitro experiments, the Ali14 mutation is thought to be a novel gain-of-function allele of Plcg2. Findings from systematic screening of Ali14/+ mice demonstrated various phenotypic changes: an abnormally high T cell:B cell ratio, up-regulation of Ig, alterations in body composition, and a reduction in cholesterol and triglyceride levels in peripheral blood. In addition, spermatozoa from Ali14/+ mice failed to fertilize eggs in vitro, despite the normal fertility of the Ali14/+ male mice in vivo. CONCLUSION: These results suggest that the Plcg2-mediated pathways play a crucial role in various metabolic and sperm functions, in addition to initiating and maintaining the immune system. These findings may indicate the importance of the Ali14/+ mouse strain as a model for systemic inflammatory diseases and inflammation-related metabolic changes in humans.

Increased inflammation and impaired resistance to Chlamydophila pneumoniae infection in Dusp1(-/-) mice: critical role of IL-6.

The MAPK phosphatase DUSP1 is an essential negative regulator of TLR-triggered innate immune activation. Here, we have investigated the impact of DUSP1 on inflammatory and antimicrobial host responses to the intracellular pathogen Chlamydophila pneumoniae. Following nasal infection, DUSP1-deficient mice mounted an enhanced pulmonary cytokine (IL-1beta, IL-6) and chemokine response (CCL3, CCL4, CXCL1, CXCL2), leading to increased leukocyte infiltration. Of interest, the increased inflammatory response, in the absence of DUSP1, was associated with higher bacterial numbers in the lungs, although the expression of IFN-gamma and critical antichlamydial effector molecules, such as iNOS, was intact. Blockade of IL-6 trans-signaling by injection of a soluble gp130-Fc fusion protein corrected the overshooting chemokine production as well as the increased chlamydial load in Dusp1(-/-) mice. Furthermore, IL-6 enhanced the replication of C. pneumoniae in embryonic fibroblasts in vitro. These data show that DUSP1 is required to achieve a balanced response to chlamydial infection and identify IL-6 as critical for amplifying inflammation and benefiting chlamydial growth through direct effects on infected cells.

A humanized version of Foxp2 affects cortico-basal ganglia circuits in mice.

It has been proposed that two amino acid substitutions in the transcription factor FOXP2 have been positively selected during human evolution due to effects on aspects of speech and language. Here, we introduce these substitutions into the endogenous Foxp2 gene of mice. Although these mice are generally healthy, they have qualitatively different ultrasonic vocalizations, decreased exploratory behavior and decreased dopamine concentrations in the brain suggesting that the humanized Foxp2 allele affects basal ganglia. In the striatum, a part of the basal ganglia affected in humans with a speech deficit due to a nonfunctional FOXP2 allele, we find that medium spiny neurons have increased dendrite lengths and increased synaptic plasticity. Since mice carrying one nonfunctional Foxp2 allele show opposite effects, this suggests that alterations in cortico-basal ganglia circuits might have been important for the evolution of speech and language in humans.

Is propylene oxide induced cell proliferation in rat nasal respiratory epithelium mediated by a severe depletion of water-soluble non-protein thiol?

Propylene oxide (PO) concentrations >or=300 ppm induced cell proliferation and tumors in rat nasal respiratory epithelium (NRE). Cell proliferation was suggested to result from depletion of glutathione (GSH) in NRE. In order to substantiate this hypothesis, cell proliferation - measured by bromodeoxyuridine incorporation into DNA of the epithelium lining middle septum, dorsal medial meatus, and medial and lateral surfaces of the nasoturbinate in transverse nasal sections taken immediately posterior to the upper incisor teeth - and water-soluble non-protein thiol (NPSH) in NRE were determined after exposing male Fischer 344 rats to 50 ppm, 100 ppm, 200 ppm, or 300 ppm PO (6 h/day, 3 days). Both parameters were also investigated after treating rats for 3 days with diethylmaleate (DEM; 2 x 250 mg/kg/day or 500 + 150 mg/kg/day) or buthionine sulfoximine (BSO; 500 mg/kg/day). Exposure to 50 ppm PO and treatment with 2 x2 50 mg/kg/day DEM resulted in NPSH levels approximating 50% and 80% of the level in untreated controls, respectively. Cell proliferation did not increase. After exposures to >or= 100 ppm PO or treatment with BSO or 500 + 150 mg/kg/day DEM, NPSH was depleted to

Systemic first-line phenotyping.

With the completion of the mouse genome sequence an essential task for biomedical sciences in the twenty-first century will be the generation and functional analysis of mouse models for every gene in the mammalian genome. More than 30,000 mutations in ES cells will be engineered and thousands of mouse disease models will become available over the coming years by the collaborative effort of the International Mouse Knockout Consortium. In order to realize the full value of the mouse models proper characterization, archiving and dissemination of mouse disease models to the research community have to be performed. Phenotyping centers (mouse clinics) provide the necessary capacity, broad expertise, equipment, and infrastructure to carry out large-scale systemic first-line phenotyping. Using the example of the German Mouse Clinic (GMC) we will introduce the reader to the different aspects of the organization of a mouse clinic and present selected methods used in first-line phenotyping.

EZH2 is a mediator of EWS/FLI1 driven tumor growth and metastasis blocking endothelial and neuro-ectodermal differentiation.

Ewing tumors (ET) are highly malignant, localized in bone or soft tissue, and are molecularly defined by ews/ets translocations. DNA microarray analysis revealed a relationship of ET to both endothelium and fetal neural crest. We identified expression of histone methyltransferase enhancer of Zeste, Drosophila, Homolog 2 (EZH2) to be increased in ET. Suppressive activity of EZH2 maintains stemness in normal and malignant cells. Here, we found EWS/FLI1 bound to the EZH2 promoter in vivo, and induced EZH2 expression in ET and mesenchymal stem cells. Down-regulation of EZH2 by RNA interference in ET suppressed oncogenic transformation by inhibiting clonogenicity in vitro. Similarly, tumor development and metastasis was suppressed in immunodeficient Rag2(-/-)gamma(C)(-/-) mice. EZH2-mediated gene silencing was shown to be dependent on histone deacetylase (HDAC) activity. Subsequent microarray analysis of EZH2 knock down, HDAC-inhibitor treatment and confirmation in independent assays revealed an undifferentiated phenotype maintained by EZH2 in ET. EZH2 regulated stemness genes such as nerve growth factor receptor (NGFR), as well as genes involved in neuroectodermal and endothelial differentiation (EMP1, EPHB2, GFAP, and GAP43). These data suggest that EZH2 might have a central role in ET pathology by shaping the oncogenicity and stem cell phenotype of this tumor.

Control of pathogenicity and disease specificity of a T-lymphomagenic gammaretrovirus by E-box motifs but not by an overlapping glucocorticoid response element.

Although transcription factors of the basic helix-loop-helix family have been shown to regulate enhancers of lymphomagenic gammaretroviruses through E-box motifs, the overlap of an E-box motif (Egre) with the glucocorticoid response element (GRE) has obscured their function in vivo. We report here that Egre, but not the GRE, affects disease induction by the murine T-lymphomagenic SL3-3 virus. Mutating all three copies of Egre prolonged the tumor latency period from 60 to 109 days. Further mutating an E-box motif (Ea/s) outside the enhancer prolonged the latency period to 180 days, suggesting that Ea/s works as a backup site for Egre. While wild-type SL3-3 and GRE and Ea/s mutants exclusively induced T-cell lymphomas with wild-type latencies mainly of the CD4(+) CD8(-) phenotype, Egre as well as the Egre and Ea/s mutants induced B-cell lymphomas and myeloid leukemia in addition to T-cell lymphomas. T-cell lymphomas induced by the two Egre mutants had the same phenotype as those induced by wild-type SL3-3, indicating the incomplete disruption of T-cell lymphomagenesis, which is in contrast to previous findings for a Runx site mutant of SL3-3. Mutating the Egre site or Egre and Ea/s triggered several tumor phenotype-associated secondary enhancer changes encompassing neighboring sites, none of which led to the regeneration of an E-box motif. Taken together, our results demonstrate a role for the E-box but not the GRE in T lymphomagenesis by SL3-3, unveil an inherent broader disease specificity of the virus, and strengthen the notion of selection for more potent enhancer variants of mutated viruses during tumor development.

Preclinical evaluation of the alpha-particle generator nuclide 225Ac for somatostatin receptor radiotherapy of neuroendocrine tumors.

PURPOSE: Peptide receptor radionuclide therapy (PRRT) using somatostatin analogues labeled with beta-particle-emitting isotopes such as 90Y or 177Lu has been a promising treatment strategy for metastasized neuroendocrine tumors. Although remission can be accomplished in a high percentage of neuroendocrine tumors, some tumors do not respond to this treatment. alpha-Emitting isotopes-such as the 10-day half-life alpha-emitting generator nuclide Actinum-225 (225Ac)-are characterized by extremely high cytotoxic activity on the cellular level, and may be superior in the treatment of neuroendocrine tumors not responding to PRRT using beta-emitting isotopes. EXPERIMENTAL DESIGN: Radiolabeling of 225Ac 1,4,7,10-tetra-azacylododecane N,N',N'',N'''-J-tetraacetic acid-Tyr3-octreotide (DOTATOC) was done at pH 5 (60 minutes at 70 degrees C) without further purification. Biodistribution in nude mice bearing AR42J rat pancreas neuroendocrine tumor xenografts were measured for up to 24 hours. Toxicity was tested by weight changes, retention variables (blood urea nitrogen and creatine), and histopathology in mice 7 months after treatment with 10 to 130 kBq (n = 4-5). Therapeutic efficacy was assessed by tumor weighing in animals treated 4 days after xenotransplantation and compared with 177Lu-DOTATOC as a reference. RESULTS: Activities up to 20 kBq had no significant toxic effects in mice. In contrast, activities higher than 30 kBq induced tubular necrosis. Biodistribution studies revealed that 225Ac-DOTATOC effectively accumulated in neuroendocrine xenograft tumors. 225Ac-DOTATOC activities were shown to be nontoxic (12-20 kBq), reduced the growth of neuroendocrine tumors, and showed improved efficacy compared with 177Lu-DOTATOC. CONCLUSIONS: 225Ac might be suitable to improve PRRT in neuroendocrine tumors.