Recombinant adeno-associated viral (rAAV) vectors are potentially powerful tools for gene therapy of CNS diseases, but their penetration into brain parenchyma is severely limited by the blood-brain barrier (BBB) and current delivery relies on invasive stereotactic injection. Here we evaluate the local, targeted delivery of rAAV vectors into the brains of mice by noninvasive, reversible, microbubble-facilitated focused ultrasound (FUS), resulting in BBB opening that can be monitored and controlled by magnetic resonance imaging (MRI). Using this method, we found that IV-administered AAV2-GFP (green fluorescence protein) with a low viral vector titer (1×109 vg/g) can successfully penetrate the BBB-opened brain regions to express GFP. We show that MRI monitoring of BBB-opening could serve as an indicator of the scale and distribution of AAV transduction. Transduction peaked at 3 weeks and neurons and astrocytes were affected. This novel, noninvasive delivery approach could significantly broaden the application of AAV-viral-vector-based genes for treatment of CNS diseases.

Interleukin-4 (IL-4) has been considered as one of the tolerogenic cytokines in many autoimmune animal models and clinical settings. Despite its role in antagonizing pathogenic Th1 responses, little is known about whether IL-4 possesses functions that affect regulatory T cells (Tregs). Tregs are specialized cells responsible for the maintenance of peripheral tolerance through their immune modulatory capabilities. Interestingly, it has been suggested that IL-4 supplement at a high concentration protects responder T cells (Tresps) from Treg-mediated immune suppression. In addition, such supplement also impedes TGF-β-induced Treg differentiation in vitro. However, these phenomena may contradict the tolerogenic role of IL-4, and the effects of IL-4 on Tregs are therefore needed to be further elucidated. In this study, we utilized IL-4 knockout (KO) mice to validate the role of IL-4 on Treg-mediated immune suppression. Although IL-4 KO and control animals harbor similar frequencies of Tregs, Tregs from IL-4 KO mice weakly suppressed autologous Tresp activation. In addition, IL-4 deprivation impaired the ability of Tregs to modulate immune response, whereas IL-4 supplementation reinforced IL-4 KO Tregs in their function in suppressing Tresps. Finally, the presence of IL-4 was associated with increased cell survival and granzyme expression of Tregs. These results suggest the essential role of IL-4 in supporting Treg-mediated immune suppression, which may benefit the development of therapeutic strategies for autoimmune diseases.

Two SDS-resistant endochitinases, designated as ASCHI53 and ASCHI61, were isolated from Aeromonas schubertii in a soil sample from southern Taiwan. MALDI-TOF mass measurement indicates the molecular weights of 53,527 for ASCHI53 and 61,202 for ASCHI61. N-terminal and internal amino acid sequences were obtained, and BLAST analysis of the sequences and MS/MS peptide sequencing showed that they were novel proteins. Degradation of chitin by these two endochitinases gave rise to hexameric chitin oligosaccharide, a compound known to have several potent biomedical functions. ASCHI53 and ASCHI61 retained, respectively, 65% and 75%, of their chitinase activity in the presence of 5% SDS and 100% of their activity in the presence of 10% b-mercaptoethanol. These results demonstrate that they are SDS-resistant endochitinases and probably have a rigid structure. V

a b s t r a c tTargeted inhibition of multidrug ABCG2 transporter is believed to improve cancer therapeutics. However, the consequences of ABCG2 inhibition have not been systematically evaluated since ABCG2 is expressed in several organs including the liver. Here, we demonstrate that ABCG2-deficient hepatocytes have increased amounts of fragmental mitochondria accompanied by disruption of mitochondrial dynamics and functions. This disruption was due to ABCG2 knockout elevating intracellular protoporphyrin IX, which led to upregulation of DRP-1-mediated mitochondrial fission. The finding that ABCG2 deficiency can generate dysfunctional mitochondria in hepatocytes raises concerns regarding the systematic use of ABCG2 inhibitor in cancer patients.