Brain arteriovenous malformation (bAVM) is an important risk factor for intracranial hemorrhage. The pathogenesis of bAVM has not been fully understood. Animal models are important tools for dissecting bAVM pathogenesis and testing new therapies. We have developed several mouse bAVM models using genetically modified mice. However, due to the body size, mouse bAVM models have some limitations. Recent studies identified somatic mutations in sporadic human bAVM. To develop a feasible tool to create sporadic bAVM in rodent and animals larger than rodent, we made tests using the CRISPR/Cas9 technique to induce somatic gene mutations in mouse brain in situ. Two sequence-specific guide RNAs (sgRNAs) targeting mouse Alk1 exons 4 and 5 were cloned into pAd-Alk1e4sgRNA + e5sgRNA-Cas9 plasmid. These sgRNAs were capable to generate mutations in Alk1 gene in mouse cell lines. After packaged into adenovirus, Ad-Alk1e4sgRNA + e5sgRNA-Cas9 was co-injected with an adeno-associated viral vector expressing vascular endothelial growth factor (AAV-VEGF) into the brains of wild-type C57BL/6J mice. Eight weeks after viral injection, bAVMs were detected in 10 of 12 mice. Compared to the control (Ad-GFP/AAV-VEGF-injected) brain, 13% of Alk1 alleles were mutated and Alk1 expression was reduced by 26% in the Ad-Alk1e4sgRNA + e5sgRNA-Cas9/AAV-VEGF-injected brains. Around the Ad-Alk1e4sgRNA + e5sgRNA-Cas9/AAV-VEGF injected site, Alk1-null endothelial cells were detected. Our data demonstrated that CRISPR/Cas9 is a feasible tool for generating bAVM model in animals.

Antifreeze proteins and glycoproteins [AF(G)Ps] have been well-known for more than three decades for their ability to inhibit the growth and recrystallization of ice through binding to specific ice crystal faces, and they show remarkable structural compatibility with specific ice crystal faces. Here, we show that the crystal growth faces of methyl α-D-mannopyranoside (MDM), a representative pyranose sugar, also show noteworthy structural compatibility with the known periodicities of AF(G)Ps. We selected fish AFGPs (AFGP8, AFGP1-5), and a beetle AFP (DAFP1) with increasing antifreeze activity as potential additives for controlling MDM crystal growth. Similar to their effects on ice growth, the AF(G)Ps can inhibit MDM crystal growth and recrystallization, and more significantly, the effectiveness for the AF(G)Ps are well correlated with their antifreeze activity. MDM crystals grown in the presence of AF(G)Ps are smaller and have better defined shapes and are of higher quality as indicated by single crystal X-ray diffraction and polarized microscopy than control crystals, but no new polymorphs of MDM were identified by single crystal X-ray diffraction, solid-state NMR, and attenuated total reflectance infrared spectroscopy. The observed changes in the average sizes of the MDM crystals can be related to the changes in the number of the MDM nuclei in the presence of the AF(G)Ps. The critical free energy change differences of the MDM nucleation in the absence and presence of the additives were calculated. These values are close to those of the ice nucleation in the presence of AF(G)Ps suggesting similar interactions are involved in the molecular recognition of MDM by the AF(G)Ps. To our knowledge this is the first report where AF(G)Ps have been used to control crystal growth of carbohydrates and on AFGPs controlling non-ice-like crystals. Our finding suggests MDM might be a possible alternative to ice for studying the detailed mechanism of AF(G)P-crystal interactions. The relationships between AF(G)Ps and carbohydrate binding proteins are also discussed. The structural compatibility between AF(G)Ps and growing crystal faces demonstrated herein adds to the repertoire of molecular recognition by AF(G)Ps, which may have potential applications in the sugar, food, pharmaceutical, and materials industries.

Purpose

Photon counting CT (PCCT) provides spectral measurements for material decomposition. However, the image noise (at a fixed dose) depends on the source spectrum. Our study investigates the potential benefits from spectral optimization using fast kV switching and filtration to reduce noise in material decomposition.

Approach

The effect of the input spectra on noise performance in both two-basis material decomposition and three-basis material decomposition was compared using Cramer-Rao lower bound analysis in the projection domain and in a digital phantom study in the image domain. The fluences of different spectra were normalized using the CT dose index to maintain constant dose levels. Four detector response models based on Si or CdTe were included in the analysis.

Results

For single kV scans, kV selection can be optimized based on the imaging task and object size. Furthermore, our results suggest that noise in material decomposition can be substantially reduced with fast kV switching. For two-material decomposition, fast kV switching reduces the standard deviation (SD) by ∼10% . For three-material decomposition, greater noise reduction in material images was found with fast kV switching (26.2% for calcium and 25.8% for iodine, in terms of SD), which suggests that challenging tasks benefit more from the richer spectral information provided by fast kV switching.

Conclusions

The performance of PCCT in material decomposition can be improved by optimizing source spectrum settings. Task-specific tube voltages can be selected for single kV scans. Also, our results demonstrate that utilizing fast kV switching can substantially reduce the noise in material decomposition for both two- and three-material decompositions, and a fixed Gd filter can further enhance such improvements for two-material decomposition.

Preventing spontaneous crystallization of supersaturated solutions by additives is of critical interest to successful process design and implementation, with numerous applications in chemical, pharmaceutical, medical, pigment, and food industries, but challenges remain in laboratory and industry settings and fundamental understanding is lacking. When copresented with antifreeze proteins (AFPs), otherwise spontaneously crystallizing osmolytes are maintained at high supersaturations for months in over-wintering organisms. Thus, we here explore the inhibition phenomenon by AFPs, using persistent crystallization of a common sugar alcohol, D-mannitol, as a case study. We report experimentally that DAFP1, an insect AFP, completely inhibits D-mannitol nucleation. Computer simulations reveal a new mechanism for crystallization inhibition where the population of the crystal-forming conformers are selectively bound and randomized in solution by hydrogen bonding to the protein surface. These results highlight the advantages of using natural polymers to address crystallization inhibition challenges and suggest new strategies in controlling the nucleation processes.

Background and purpose

Brain arteriovenous malformation (bAVM) is an important risk factor for intracranial hemorrhage. Current treatments for bAVM are all associated with considerable risks. There is no safe method to prevent bAVM hemorrhage. Thalidomide reduces nose bleeding in patients with hereditary hemorrhagic telangiectasia, an inherited disorder characterized by vascular malformations. In this study, we tested whether thalidomide and its less toxic analog, lenalidomide, reduce bAVM hemorrhage using a mouse model.

Methods

bAVMs were induced through induction of brain focal activin-like kinase 1 (Alk1, an AVM causative gene) gene deletion and angiogenesis in adult Alk1-floxed mice. Thalidomide was injected intraperitoneally twice per week for 6 weeks, starting either 2 or 8 weeks after AVM induction. Lenalidomide was injected intraperitoneally daily starting 8 weeks after AVM induction for 6 weeks. Brain samples were collected at the end of the treatments for morphology, mRNA, and protein analyses. The influence of Alk1 downregulation on PDGFB (platelet-derived growth factor B) expression was also studied on cultured human brain microvascular endothelial cells. The effect of PDGFB in mural cell recruitment in bAVM was explored by injection of a PDGFB overexpressing lentiviral vector to the mouse brain.

Results

Thalidomide or lenalidomide treatment reduced the number of dysplastic vessels and hemorrhage and increased mural cell (vascular smooth muscle cells and pericytes) coverage in the bAVM lesion. Thalidomide reduced the burden of CD68⁺ cells and the expression of inflammatory cytokines in the bAVM lesions. PDGFB expression was reduced in ALK1-knockdown human brain microvascular endothelial cells and in mouse bAVM lesion. Thalidomide increased Pdgfb expression in bAVM lesion. Overexpression of PDGFB mimicked the effect of thalidomide.

Conclusions

Thalidomide and lenalidomide improve mural cell coverage of bAVM vessels and reduce bAVM hemorrhage, which is likely through upregulation of Pdgfb expression.