ObjectiveMutations in the AIFM1 gene have been identified in recessive X-linked mitochondrial diseases. Functional and molecular consequences of these pathogenic AIFM1 mutations have been poorly studied in vivo.Methods/resultsHere we provide evidence that the disease-associated apoptosis-inducing factor (AIF) deletion arginine 201 (R200 in rodents) causes pathology in knockin mice. Within a few months, posttranslational loss of the mutant AIF protein induces severe myopathy associated with a lower number of cytochrome c oxidase-positive muscle fibers. At a later stage, Aifm1 (R200 del) knockin mice manifest peripheral neuropathy, but they do not show neurodegenerative processes in the cerebellum, as observed in age-matched hypomorphic Harlequin (Hq) mutant mice. Quantitative proteomic and biochemical data highlight common molecular signatures of mitochondrial diseases, including aberrant folate-driven one-carbon metabolism and sustained Akt/mTOR signaling.ConclusionOur findings indicate metabolic defects and distinct tissue-specific vulnerability due to a disease-causing AIFM1 mutation, with many pathological hallmarks that resemble those seen in patients.

Micelle formulated ICG can be visualized in the retinal vasculature and laser-induced CNV in vivo and ex vivo. Micellar ICG/HS 15 showed in vivo stronger signal intensity when compared to ICG for all tested dosages. Following further investigations, ICG/HS 15 may be evaluated in patients with retinal and choroidal diseases for more refined diagnosis.

Pharmacokinetics of fluorescent-labeled bevacizumab, B20-4.1.1 and AF564 can be investigated in vivo. In this model, interpretation of long-term in vivo observations is difficult because of a possible rat-specific immune response and challenges to image localized binding of soluble VEGF. Further investigations in a primate model and the use of appropriate antibodies directed against the VEGF-receptor may represent alternative approaches.

Incubation of β‐escin‐permeabilized guinea‐pig longitudinal ileal smooth muscle with ATPγS under conditions that do not lead to thiophosphorylation of regulatory light chains of myosin (r‐MLC) increased subsequent Ca2+ sensitivity of force and r‐MLC phosphorylation. In this study we tested whether this is due to activation of the Rho and/or Rho‐associated kinase (ROK) as it is the case in agonist‐induced Ca2+ sensitization. The increase in Ca2+ sensitivity induced by pretreatment with ATPγS at pCa > 8 with the myosin light chain kinase (MLCK) inhibitor ML‐9 in rigor solution was associated with 35S incorporation into the regulatory subunit of myosin light chain phosphatase (MLCP), MYPT1, and several other high molecular mass proteins. No thiophosphorylation of r‐MLC, MLCK, caldesmon, calponin and CPI‐17 was detected. While the relatively specific inhibitor of ROK, Y 27632, inhibited the carbachol‐induced increase in Ca2+ sensitivity with an IC50 of 1.4 μM, the ATPγS‐induced increase in Ca2+ sensitivity and thiophosphorylation of MYPT1 was not inhibited. Inhibiton of Rho by exoenzyme C3 also had no effect. Only staurosporine (2 μM), but not the PKC inhibitor peptide 19‐31, nor genistein nor PD 98059, inhibited the ATPγS‐induced Ca2+ sensitization of force, r‐MLC phosphorylation, and the 35S incorporation into MYPT1. The staurosporine‐sensitive kinase(s) appeared to be tightly associated with the contractile apparatus because treatment of Triton‐skinned preparations with ATPγS also induced a staurosporine‐sensitive increase in Ca2+ sensitivity of contraction. Since there was very little immunoreactivity with antibodies to p21‐associated kinase (PAK) in Triton‐skinned preparations, the staurosporine‐sensitive kinase most probably is not PAK. GTPγS had an additive effect on ATPγS‐induced sensitization at saturating concentrations of ATPγS. The additional effect of GTPγS was inhibited by Y 27632. We conclude that treatment with ATPγS under ATP‐free conditions, unmasks a staurosporine‐sensitive kinase which induces a large increase in Ca2+ sensitivity that is most likely to be due to thiophosphorylation of MYPT1. The kinase is distinct from ROK. The physiological significance of this kinase, which is tightly associated with the contractile apparatus, is not known at present.

Fibrillin-1 assembles into microfibrils that not only define the structural integrity and biomechanics of the aorta but also target and sequester growth factors within the extracellular microenvironment of aortic resident cells. To better understand how dominant negative effects on fibrillin microfibril stability manifest in growth factor driven aortic disease, we analyzed early events of aortic aneurysm formation within the first two weeks of postnatal life in the dominant negative Fbn1 GT8 Marfan mouse model. Echocardiography analysis of homozygous GT8 Fbn1 mice showed significant aortic root enlargement within the second week of postnatal life which correlated with the onset of fibrillin-1 fiber degradation, aberrantly increased BMP activity and upregulated transcript levels of the collagenase MMP-13. We also found the aortic collagen network structurally disturbed where the mutant GT8-fibrillin-1 was detected. Genetic ablation or pharmacological inhibition of MMP-13 in Fbn1 GT8 Marfan mice prevents aortic root dilatation implicating the relevance of this mechanism in aortic aneurysm formation in Marfan syndrome.