Safe and effective in vivo delivery of DNA and RNA using proteolipid vehicles.

Genetic medicines show promise for treating various diseases, yet clinical success has been limited by tolerability, scalability, and immunogenicity issues of current delivery platforms. To overcome these, we developed a proteolipid vehicle (PLV) by combining features from viral and non-viral approaches. PLVs incorporate fusion-associated small transmembrane (FAST) proteins isolated from fusogenic orthoreoviruses into a well-tolerated lipid formulation, using scalable microfluidic mixing. Screening a FAST protein library, we identified a chimeric FAST protein with enhanced membrane fusion activity that improved gene expression from an optimized lipid formulation. Systemically administered FAST-PLVs showed broad biodistribution and effective mRNA and DNA delivery in mouse and non-human primate models. FAST-PLVs show low immunogenicity and maintain activity upon repeat dosing. Systemic administration of follistatin DNA gene therapy with FAST-PLVs raised circulating follistatin levels and significantly increased muscle mass and grip strength. These results demonstrate the promising potential of FAST-PLVs for redosable gene therapies and genetic medicines.

Extracellular fluid viscosity enhances cell migration and cancer dissemination.

Cells respond to physical stimuli, such as stiffness1, fluid shear stress2 and hydraulic pressure3,4. Extracellular fluid viscosity is a key physical cue that varies under physiological and pathological conditions, such as cancer5. However, its influence on cancer biology and the mechanism by which cells sense and respond to changes in viscosity are unknown. Here we demonstrate that elevated viscosity counterintuitively increases the motility of various cell types on two-dimensional surfaces and in confinement, and increases cell dissemination from three-dimensional tumour spheroids. Increased mechanical loading imposed by elevated viscosity induces an actin-related protein 2/3 (ARP2/3)-complex-dependent dense actin network, which enhances Na+/H+ exchanger 1 (NHE1) polarization through its actin-binding partner ezrin. NHE1 promotes cell swelling and increased membrane tension, which, in turn, activates transient receptor potential cation vanilloid 4 (TRPV4) and mediates calcium influx, leading to increased RHOA-dependent cell contractility. The coordinated action of actin remodelling/dynamics, NHE1-mediated swelling and RHOA-based contractility facilitates enhanced motility at elevated viscosities. Breast cancer cells pre-exposed to elevated viscosity acquire TRPV4-dependent mechanical memory through transcriptional control of the Hippo pathway, leading to increased migration in zebrafish, extravasation in chick embryos and lung colonization in mice. Cumulatively, extracellular viscosity is a physical cue that regulates both short- and long-term cellular processes with pathophysiological relevance to cancer biology.

Intravital imaging of Wnt/ß-catenin and ATF2-dependent signalling pathways during tumour cell invasion and metastasis.

Wnt signalling has been implicated as a driver of tumour cell metastasis, but less is known about which branches of Wnt signalling are involved and when they act in the metastatic cascade. Here, using a unique intravital imaging platform and fluorescent reporters, we visualised ß-catenin/TCF-dependent and ATF2-dependent signalling activities during human cancer cell invasion, intravasation and metastatic lesion formation in the chick embryo host. We found that cancer cells readily shifted between states of low and high canonical Wnt activity. Cancer cells that displayed low Wnt canonical activity showed higher invasion and intravasation potential in primary tumours and in metastatic lesions. In contrast, cancer cells showing low ATF2-dependent activity were significantly less invasive both at the front of primary tumours and in metastatic lesions. Simultaneous visualisation of both these reporters using a double-reporter cell line confirmed their complementary activities in primary tumours and metastatic lesions. These findings might inform the development of therapies that target different branches of Wnt signalling at specific stages of metastasis.

Atypical co-development of the thalamus and cortex in autism: Evidence from age-related white-gray contrast change.

Recent studies have shown that white-gray contrast (WGC) of either cortical or subcortical gray matter provides for accurate predictions of age in typically developing (TD) children, and that, at least for the cortex, it changes differently with age in subjects with autism spectrum disorder (ASD) compared to their TD peers. Our previous study showed different patterns of contrast change between ASD and TD in sensorimotor and association cortices. While that study was confined to the cortex, we hypothesized that subcortical structures, particularly the thalamus, were involved in the observed cortical dichotomy between lower and higher processing. The current paper investigates that hypothesis using the WGC measures from the thalamus in addition to those from the cortex. We compared age-related WGC changes in the thalamus to those in the cortex. To capture the simultaneity of this change across the two structures, we devised a metric capturing the co-development of the thalamus and cortex (CoDevTC), proportional to the magnitude of cortical and thalamic age-related WGC change. We calculated this metric for each of the subjects in a large homogeneous sample taken from the Autism Brain Imaging Data Exchange (ABIDE) (N = 434). We used structural MRI data from the largest high-quality cross-sectional sample (NYU) as well as two other large high-quality sites, GU and OHSU, all three using Siemens 3T scanners. We observed that the co-development features in ASD and TD exhibit contrasting patterns; specifically, some higher-order thalamic nuclei, such as the lateral dorsal nucleus, exhibited reduction in codevelopment with most of the cortex in ASD compared to TD. Moreover, this difference in the CoDevTC pattern correlates with a number of behavioral measures across multiple cognitive and physiological domains. The results support previous notions of altered connectivity in autism, but add more specific evidence about the heterogeneity in thalamocortical development that elucidates the mechanisms underlying the clinical features of ASD.

Associations between maternal pre-pregnancy BMI and infant striatal mean diffusivity.

BACKGROUND: It is well-established that parental obesity is a strong risk factor for offspring obesity. Further, a converging body of evidence now suggests that maternal weight profiles may affect the developing offspring's brain in a manner that confers future obesity risk. Here, we investigated how pre-pregnancy maternal weight status influences the reward-related striatal areas of the offspring's brain during in utero development. METHODS: We used diffusion tensor imaging to quantify the microstructure of the striatal brain regions of interest in neonates (N = 116 [66 males, 50 females], mean gestational weeks at birth [39.88], SD = 1.14; at scan [43.56], SD = 1.05). Linear regression was used to test the associations between maternal pre-pregnancy body mass index (BMI) and infant striatal mean diffusivity. RESULTS: High maternal pre-pregnancy BMI was associated with higher mean MD values in the infant's left caudate nucleus. Results remained unchanged after the adjustment for covariates. CONCLUSIONS: In utero exposure to maternal adiposity might have a growth-impairing impact on the mean diffusivity of the infant's left caudate nucleus. Considering the involvement of the caudate nucleus in regulating eating behavior and food-related reward processing later in life, this finding calls for further investigations to define the prognostic relevance of early-life caudate nucleus development and weight trajectories of the offspring.

Effect of number of diffusion encoding directions in neonatal diffusion tensor imaging using Tract-Based Spatial Statistical analysis.

Diffusion tensor imaging (DTI) has been used to study the developing brain in early childhood, infants and in utero studies. In infants, number of used diffusion encoding directions has traditionally been smaller in earlier studies down to the minimum of 6 orthogonal directions. Whereas the more recent studies often involve more directions, number of used directions remain an issue when acquisition time is optimized without compromising on data quality and in retrospective studies. Variability in the number of used directions may introduce bias and uncertainties to the DTI scalar estimates that affect cross-sectional and longitudinal study of the brain. We analysed DTI images of 133 neonates, each data having 54 directions after quality control, to evaluate the effect of number of diffusion weighting directions from 6 to 54 with interval of 6 to the DTI scalars with Tract-Based Spatial Statistics (TBSS) analysis. The TBSS analysis was applied to DTI scalar maps, and the mean region of interest (ROI) values were extracted using JHU atlas. We found significant bias in ROI mean values when only 6 directions were used (positive in fractional anisotropy [FA] and negative in fractional anisotropy [MD], axial diffusivity [AD] and fractional anisotropy [RD]), while when using 24 directions and above, the difference to scalar values calculated from 54 direction DTI was negligible. In repeated measures voxel-wise analysis, notable differences to 54 direction DTI were observed with 6, 12 and 18 directions. DTI measurements from data with at least 24 directions may be used in comparisons with DTI measurements from data with higher numbers of directions.

Prenatal maternal depressive symptoms are associated with neonatal left amygdala microstructure in a sex-dependent way.

Exposures to prenatal maternal depressive symptoms (PMDS) may lead to neurodevelopmental changes in the offspring in a sex-dependent way. Although a connection between PMDS and infant brain development has been established by earlier studies, the relationship between PMDS exposures measured at various prenatal stages and microstructural alterations in fundamental subcortical structures such as the amygdala remains unknown. In this study, we investigated the associations between PMDS measured during gestational weeks 14, 24 and 34 and infant amygdala microstructural properties using diffusion tensor imaging. We explored amygdala mean diffusivity (MD) alterations in response to PMDS in infants aged 11 to 54 days from birth. PMDS had no significant main effect on the amygdala MD metrics. However, there was a significant interaction effect for PMDS and infant sex in the left amygdala MD. Compared with girls, boys exposed to greater PMDS during gestational week 14 showed significantly higher left amygdala MD. These results indicate that PMDS are linked to infants' amygdala microstructure in boys. These associations may be relevant to later neuropsychiatric outcomes in the offspring. Further research is required to better understand the mechanisms underlying these associations and to develop effective interventions to counteract any potential adverse consequences.

Bloody noise: The impact of blood-flow artifacts on registration.

Blood-flow artifacts present a serious challenge for most, if not all, volumetric analytical approaches. We utilize T1-weighted data with prominent blood-flow artifacts from the Autism Brain Imaging Data Exchange (ABIDE) multisite agglomerative dataset to assess the impact that such blood-flow artifacts have on registration of T1-weighted data to a template. We use a heuristic approach to identify the blood-flow artifacts in these data; we use the resulting blood masks to turn the underlying voxels to the intensity of the cerebro-spinal fluid, thus mimicking the effect of blood suppression. We then register both the original data and the deblooded data to a common T1-weighted template, and compare the quality of those registrations to the template in terms of similarity to the template. The registrations to the template based on the deblooded data yield significantly higher similarity values compared with those based on the original data. Additionally, we measure the nonlinear deformations needed to transform the data from the position achieved by registering the original data to the template to the position achieved by registering the deblooded data to the template. The results indicate that blood-flow artifacts may seriously impact data processing that depends on registration to a template, that is, most all data processing.

Sex differences, asymmetry, and age-related white matter development in infants and 5-year-olds as assessed with tract-based spatial statistics.

The rapid white matter (WM) maturation of first years of life is followed by slower yet long-lasting development, accompanied by learning of more elaborate skills. By the age of 5 years, behavioural and cognitive differences between females and males, and functions associated with brain lateralization such as language skills are appearing. Diffusion tensor imaging (DTI) can be used to quantify fractional anisotropy (FA) within the WM and increasing values correspond to advancing brain development. To investigate the normal features of WM development during early childhood, we gathered a DTI data set of 166 healthy infants (mean 3.8 wk, range 2-5 wk; 89 males; born on gestational week 36 or later) and 144 healthy children (mean 5.4 years, range 5.1-5.8 years; 76 males). The sex differences, lateralization patterns and age-dependent changes were examined using tract-based spatial statistics (TBSS). In 5-year-olds, females showed higher FA in wide-spread regions in the posterior and the temporal WM and more so in the right hemisphere, while sex differences were not detected in infants. Gestational age showed stronger association with FA values compared to age after birth in infants. Additionally, child age at scan associated positively with FA around the age of 5 years in the body of corpus callosum, the connections of which are important especially for sensory and motor functions. Lastly, asymmetry of WM microstructure was detected already in infants, yet significant changes in lateralization pattern seem to occur during early childhood, and in 5-year-olds the pattern already resembles adult-like WM asymmetry.

Antibody titrations are critical for microflow cytometric analysis of extracellular vesicles.

Optimization of flow cytometry assays for extracellular vesicles (EVs) often fail to include appropriate reagent titrations - the most critically antibody titration is either not performed or is incomplete. Using nonoptimal antibody concentration is one of the main sources of error leading to a lack of reproducible data. Antibody titration for the analysis of antigens on the surface of EVs is challenging for a variety of technical reasons. Using platelets as surrogates for cells and platelet-derived particles as surrogates for EV populations, we demonstrate our process for antibody titration, highlighting some of the key analysis parameters that may confound and surprise new researchers moving into the field of EV research. Additional care must be exercised to ensure instrument and reagent controls are utilized appropriately. Complete graphical analysis of positive and negative signal intensities, concentration, and separation or stain index data is highly beneficial when paired with visual analysis of the cytometry data. Using analytical flow cytometry procedures optimized for cells for EV analysis can lead to misleading and nonreproducible results.