Brain microvasculature defects and Glut1 deficiency syndrome averted by early repletion of the glucose transporter-1 protein

Haploinsufficiency of the SLC2A1 gene and paucity of its translated product, the glucose transporter-1 (Glut1) protein, disrupt brain function and cause the neurodevelopmental disorder, Glut1 deficiency syndrome (Glut1 DS). There is little to suggest how reduced Glut1 causes cognitive dysfunction and no optimal treatment for Glut1 DS. We used model mice to demonstrate that low Glut1 protein arrests cerebral angiogenesis, resulting in a profound diminution of the brain microvasculature without compromising the blood-brain barrier. Studies to define the temporal requirements for Glut1 reveal that pre-symptomatic, AAV9-mediated repletion of the protein averts brain microvasculature defects and prevents disease, whereas augmenting the protein late, during adulthood, is devoid of benefit. Still, treatment following symptom onset can be effective; Glut1 repletion in early-symptomatic mutants that have experienced sustained periods of low brain glucose nevertheless restores the cerebral microvasculature and ameliorates disease. Timely Glut1 repletion may thus constitute an effective treatment for Glut1 DS

Brain microvasculature defects and Glut1 deficiency syndrome averted by early repletion of the glucose transporter-1 protein

Haploinsufficiency of the SLC2A1 gene and paucity of its translated product, the glucose transporter-1 (Glut1) protein, disrupt brain function and cause the neurodevelopmental disorder, Glut1 deficiency syndrome (Glut1 DS). There is little to suggest how reduced Glut1 causes cognitive dysfunction and no optimal treatment for Glut1 DS. We used model mice to demonstrate that low Glut1 protein arrests cerebral angiogenesis, resulting in a profound diminution of the brain microvasculature without compromising the blood–brain barrier. Studies to define the temporal requirements for Glut1 reveal that pre-symptomatic, AAV9-mediated repletion of the protein averts brain microvasculature defects and prevents disease, whereas augmenting the protein late, during adulthood, is devoid of benefit. Still, treatment following symptom onset can be effective; Glut1 repletion in early-symptomatic mutants that have experienced sustained periods of low brain glucose nevertheless restores the cerebral microvasculature and ameliorates disease. Timely Glut1 repletion may thus constitute an effective treatment for Glut1 DS

Structural and optical properties of penicillamine-protected gold nanocluster fractions separated by sequential size-selective fractionation

Polydisperse water-soluble gold nanoclusters (AuNCs) protected by penicillamine have been synthesized in this work. The sequential size-selective precipitation (SSSP) technique has been applied for the size fractionation and purification of the monolayer-protected AuNCs. Through continuously adding acetone to a crude AuNC aqueous solution and controlling the volume percentage of acetone, we successfully separated the polydisperse AuNCs with diameters ranging from 0.5 to 5.4 nm into four different fractions sequentially. High-resolution transmission electron microscopy (HRTEM) shows that the four fractions are well-dispersed spherical particles of diameter 3.0 ± 0.6, 2.3 ± 0.5, 1.7 ± 0.4, and 1.2 ± 0.4 nm. Proton nuclear magnetic resonance spectroscopy suggests that disulfide, excess ligands and gold(I) complexes were removed from the AuNCs fractions. These results demonstrate the considerable potential of the SSSP technique for size-based separation and purification of AuNCs, achieving not only the isolation of larger nanoclusters (NCs) from small NCs in a continuous fashion, but also for the removal of small-molecule impurities. Based on the results from the mass spectrometry and thermogravimetric analysis, the average composition of the four fractions can be represented by Au38(SR)18, Au28(SR)15, Au18(SR)12, and Au11(SR)8, respectively. This indicates that the SSSP separation is mainly dependent on the core size and the ratio of Au atoms to ligands of AuNCs. X-ray photoelectron spectroscopy (XPS) has also been applied to observe the molecular dependence on the gold and sulfur chemical state of organosulfur monolayers of the fractions. The photoluminescence spectra of these AuNCs in the range of 900–790 nm was investigated at room temperature. The results show that the peak emission energy of the size-selected AuNCs undergoes a blue shift when the size is decreased, which can be attributed to the quantum confinement effect

Safety Evaluation of Neo Transgenic Pigs by Studying Changes in Gut Microbiota Using High-Throughput Sequencing Technology.

The neo (neomycin phosphotransferase) gene is widely used as a selection marker in the production of genetically engineered animals and plants. Recent attention has been focused on safety concerns regarding neo transgene expression. In this study, neo transgenic and non-transgenic piglets were randomly assigned into Group A and Group B to evaluate effects of neo transgene by studying changes in gut microbiota using high-throughput sequencing. Group A pigs were fed a standard diet supplemented with antibiotic neomycin; Group B pigs were fed a standard diet. We examined horizontal transfer of exogenous neo gene using multiplex PCR; and investigated if the presence of secreted NPT II (neo expression product) in the intestine could lead to some protection against neomycin in transgenic pigs by monitoring different patterns of changes in gut microbiota in Group A animals. The unintended effects of neo transgene on gut microbiota were studied in Group B animals. Horizontal gene transfer was not detected in gut microbiota of any transgenic pigs. In Group A, a significant difference was observed between transgenic pigs and non-transgenic pigs in pattern of changes in Proteobacteria populations in fecal samples during and post neomycin feeding. In Group B, there were significant differences in the relative abundance of phyla Firmicutes, Bacteroidetes and Proteobacteria, and genera Lactobacillus and Escherichia-Shigella-Hafnia between transgenic pigs and non-transgenic pigs. We speculate that the secretion of NPT II from transgenic tissues/cells into gut microbiota results in the inhibition of neomycin activity and the different patterns of changes in bacterial populations. Furthermore, the neo gene also leads to unintended effects on gut microbiota in transgenic pigs that were fed with basic diet (not supplemented with neomycin). Thus, our data in this study caution that wide use of the neo transgene in genetically engineered animals should be carefully considered and fully assessed

<i>Neo</i> transgene integration and expression.

<p>a: <i>Neo</i> plasmid PEZ1.1. b: PCR detection of <i>neo</i> transgene integration in Group A and Group B pigs. The PCR product at 666 bp indicates the integration of <i>neo</i> in transgenic pigs (lanes 1–4 and 9–13). No PCR product was detected in non-transgenic pigs (lanes 5–8 and 14–18). c. Southern blot of transgenic (lanes 6–9 and 14–18) and non-transgenic (lanes 2–5 and 11–13) pig samples from Groups A and B. 3760 bp is the length of DNA containing the <i>neo</i> gene. d. Western blot of intestinal tissue extracts from transgenic pigs (lanes 1–4 and 7–11) and non-transgenic (lanes 5–6 and 12–13) pigs. Note that the <i>neo</i> expression product NPT II was detected as a protein band at 29 kDa in transgenic samples only. The GAPDH band at 37 kDa is the internal reference.</p

PCR detection of horizontal transfer of the <i>neo</i> transgene to the gut microbiota of transgenic pigs.

<p>a: Samples from Group A pigs: lane 1, <i>neo</i> positive control plasmid; lane 2, ear sample from a transgenic pig; lane 3, ear sample from a non-transgenic pig; lane 4, blank; lanes 5–8, fecal samples from transgenic pigs collected at day 0, day 8, day 23 and day 45 post antibiotic feeding; lanes 9–12, fecal samples from non-transgenic pigs collected at day 0, day 8, day 23 and day 45 post antibiotic feeding. b: Samples from group B pigs: lane 13, <i>neo</i> positive control plasmid; lane 14, ear sample from a transgenic pig; lane 15, ear sample from a non-transgenic pig; lane 16, blank; lanes 17–22, fecal samples from the duodenum, jejunum, ileum, cecum, colon, and rectum of transgenic pigs; lanes 23–28, fecal samples from the duodenum, jejunum, ileum, cecum, colon, and rectum of non-transgenic pigs. M: molecular weight markers. 845 bp: PCR product of <i>neo</i> gene and SV40 promoter (primers P3 and P4); 396 bp: PCR product of the porcine beta-actin gene (primers P5 and P6); 460 bp: PCR product of 16S rDNA (primers P7 and P8).</p

An early endothelial cell–specific requirement for Glut1 is revealed in Glut1 deficiency syndrome model mice

Paucity of the glucose transporter-1 (Glut1) protein resulting from haploinsufficiency of the SLC2A1 gene arrests cerebral angiogenesis and disrupts brain function to cause Glut1 deficiency syndrome (Glut1 DS). Restoring Glut1 to Glut1 DS model mice prevents disease, but the precise cellular sites of action of the transporter, its temporal requirements, and the mechanisms linking scarcity of the protein to brain cell dysfunction remain poorly understood. Here, we show that Glut1 functions in a cell-autonomous manner in the cerebral microvasculature to affect endothelial tip cells and, thus, brain angiogenesis. Moreover, brain endothelial cell–specific Glut1 depletion not only triggers a severe neuroinflammatory response in the Glut1 DS brain, but also reduces levels of brain-derived neurotrophic factor (BDNF) and causes overt disease. Reduced BDNF correlated with fewer neurons in the Glut1 DS brain. Controlled depletion of the protein demonstrated that brain pathology and disease severity was greatest when Glut1 scarcity was induced neonatally, during brain angiogenesis. Reducing Glut1 at later stages had mild or little effect. Our results suggest that targeting brain endothelial cells during early development is important to ensure proper brain angiogenesis, prevent neuroinflammation, maintain BDNF levels, and preserve neuron numbers. This requirement will be essential for any disease-modifying therapeutic strategy for Glut1 DS

Different patterns in changes in genus level between transgenic and non-transgenic pigs post neomycin feedings in Group A.

<p>Comparison of changes in the levels of <i>Lactobacillus</i> (a) and genus <i>Prevotella</i> (b) at various time points (0D, 8D, 23 and 45D) post neomycin feeding between transgenic (T) and non-transgenic (NT) pigs. *: P<0.05.</p