Altered neural connectivity in females, but not males with autism: preliminary evidence for the female protective effect from a quality-controlled diffusion tensor imaging study

Previous studies using diffusion tensor imaging (DTI) to investigate white matter (WM) structural connectivity have suggested widespread, although inconsistent WM alterations in autism spectrum disorder (ASD), such as greater reductions in fractional anisotropy (FA). However, findings may lack generalizability because: (a) most have focused solely on the ASD male brain phenotype, and not sex-differences in WM integrity; (b) many lack stringent and transparent data quality control such as controlling for head motion in analysis. This study addressed both issues by using Tract-Based Spatial Statistics (TBSS) to separately compare WM differences in 81 ASD (56 male, 25 female; 4–21 years old) and 39 typically developing (TD; 23 males, 16 females; 5–18 years old) children and young people, carefully group-matched on sex, age, cognitive abilities, and head motion. ASD males and females were also matched on autism symptom severity. Two independent-raters completed a multistep scan quality assurance to remove images that were significantly distorted by motion artifacts before analysis. ASD females exhibited significant widespread reductions in FA compared to TD females, suggesting altered WM integrity. In contrast, no significant localized or widespread WM differences were found between ASD and TD males. This study highlights the importance of data quality control in DTI, and outlines important sex-differences in WM alterations in ASD females. Future studies can explore the extent to which neural structural differences might underlie sex-differences in ASD behavioral phenotype, and guide clinical interventions to be tailored toward the unique needs of ASD females and males

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. However, it is still unknown whether stem and progenitor cells exist in the adult pancreas. Research strategies using cre-lox lineage-tracing in adult mice have yielded results that either support or refute the idea that beta cells can be generated from the ducts, the presumed location where adult pancreatic progenitors may reside. These in vivo cre-lox lineage-tracing methods, however, cannot answer the questions of self-renewal and multi-lineage differentiation-two criteria necessary to define a stem cell. To begin addressing this technical gap, we devised 3-dimensional colony assays for pancreatic progenitors. Soon after our initial publication, other laboratories independently developed a similar, but not identical, method called the organoid assay. Compared to the organoid assay, our method employs methylcellulose, which forms viscous solutions that allow the inclusion of extracellular matrix proteins at low concentrations. The methylcellulose-containing assays permit easier detection and analyses of progenitor cells at the single-cell level, which are critical when progenitors constitute a small sub-population, as is the case for many adult organ stem cells. Together, results from several laboratories demonstrate in vitro self-renewal and multi-lineage differentiation of pancreatic progenitor-like cells from mice. The current protocols describe two methylcellulose-based colony assays to characterize mouse pancreatic progenitors; one contains a commercial preparation of murine extracellular matrix proteins and the other an artificial extracellular matrix protein known as a laminin hydrogel. The techniques shown here are 1) dissociation of the pancreas and sorting of CD133(+)Sox9/EGFP(+) ductal cells from adult mice, 2) single cell manipulation of the sorted cells, 3) single colony analyses using microfluidic qRT-PCR and whole-mount immunostaining, and 4) dissociation of primary colonies into single-cell suspensions and re-plating into secondary colony assays to assess self-renewal or differentiation

ALS-associated missense and nonsense TBK1 mutations can both cause loss of kinase function

Mutations in TBK1 have been linked to amyotrophic lateral sclerosis (ALS). Some TBK1 variants are nonsense and are predicted to cause disease through haploinsufficiency, however many other mutations are missense with unknown functional effect. We exome sequenced 699 familial ALS patients and identified 16 TBK1 novel or extremely rare protein changing variants. We characterised a subset of these: p.G217R, p.R357X and p.C471Y. Here we show that the p.R357X and p.G217R both abolish the ability of TBK1 to phosphorylate two of its kinase targets, IRF3 and OPTN and to undergo phosphorylation. They both inhibit binding to OPTN and the p.G217R, within the TBK1 kinase domain, reduces homodimerisation, essential for TBK1 activation and function. Lastly, we show that the proportion TBK1 that is active (phosphorylated) is reduced in five lymphoblastoid cell lines derived from patients harbouring heterozygous missense or in-frame deletion TBK1 mutations. We conclude that missense mutations in functional domains of TBK1 impair the binding and phosphorylation of its normal targets, implicating a common loss of function mechanism, analogous to truncation mutations

Organ masses.

<p>Data are presented as means±SEM. N = 6 for all the groups except for the EN group at week 19 where N = 5. Within each period, different letters indicate significant differences between treatments (P<0.05).</p

Ad libitum energy intake of male C57BL/6 mice from each diet group.

<p>The 5 diet groups: chow diet (C), high fat diet (HF), high fat diet plus Ensure® (EN), high fat diet for 7 wk followed by a switch to chow (HF-C), and high fat diet plus Ensure® for 7 wk followed by a switch to chow (EN-C). Panel A shows the energy intake derived from solid food (open bars) versus liquid Ensure (filled bars). Panel B shows the macronutrient content of the diets indicating the protein intake as open bars, carbohydrate intake as striped bars and fat intake as solid bars. Values are mean±SEM. Statistical differences among the means were determined by performing an analysis of variance. Pairwise differences were adjusted for multiple comparisons by Tukey's method. Different letters indicate significant differences of total energy intake rate across groups within each time period (P<0.05). Different numbers indicate significant within-group differences of total energy intake rate across the time periods (P<0.05).</p

Fat pad masses.

<p>Abbreviation: BAT: brown adipose tissue, MES: mesenteric fat pad, RET: retroperitoneal fat pads, EPI: epididymal fat pads, ING: inguinal fat pads. N = 6 for all the groups except for the EN group at week 19 where N = 5. Values are means±SEM. Within each period, different letters indicate significant differences between diet groups (P<0.05).</p

Energy intake, energy output, change of body energy, and ambulatory activity.

<p>Body energy changes versus cumulative energy intake over the 19 week experiment (Panel A) and the calculated 19 week average energy output rate versus average energy intake rate (Panel B). The data points indicate individual mice from the 5 diet groups: chow diet (C), high fat diet (HF), high fat diet plus Ensure® (EN), high fat diet for 7 wk followed by a switch to chow (HF-C), and high fat diet plus Ensure® for 7 wk followed by a switch to chow (EN-C). In panel B, the dotted line is the line of identity indicating average energy balance. The solid line is the best-fit regression line for the C, HF-C, and EN-C groups (slope = 0.90±0.03, intercept = 0.97±0.44 kcal/d, R2 = 0.98) and the dashed line is the best-fit regression line for the HF and EN groups (slope = 0.92±0.02, intercept = −0.21±0.28 kcal/d, R2 = 0.99). C) The average ambulatory activity of the mice measured at weeks 7 and 19. The bars with different letters indicate significant differences of ambulatory activity within each time period (P<0.05). Panel D plots the energy intake rate over the last two weeks of the study as a function of the final body weight (slope = 0.21±0.02 kcal/g/d, intercept = 4.99±0.93 kcal/d, R2 = 0.67). The regression analysis was performed using the least square method and other statistical analyses were performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005370#pone-0005370-g002" target="_blank">Figure 2</a>.</p

Body weight and body composition in male C57BL/6 mice from each group.

<p>Body weight (Panel A), fat mass (Panel B), fat free mass (Panel C), and percent body fat (Panel D) in male C57BL/6 mice from 5 diet groups: chow diet (C), high fat diet (HF), high fat diet plus Ensure® (EN), high fat diet for 7 wk followed by a switch to chow (HF-C), and high fat diet plus Ensure® for 7 wk followed by a switch to chow (EN-C). Values are mean±SEM and the error bars were often smaller than the size of the data point marker.</p