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Hereditary spastic paraplegias (HSP) are rare, inherited neurodegenerative or neurodevelopmental disorders that mainly present with lower limb spasticity and muscle weakness due to motor neuron dysfunction. Whole genome sequencing identified bi-allelic truncating variants in AMFR, encoding a RING-H2 finger E3 ubiquitin ligase anchored at the membrane of the endoplasmic reticulum (ER), in two previously genetically unexplained HSP-affected siblings. Subsequently, international collaboration recognized additional HSP-affected individuals with similar bi-allelic truncating AMFR variants, resulting in a cohort of 20 individuals from 8 unrelated, consanguineous families. Variants segregated with a phenotype of mainly pure but also complex HSP consisting of global developmental delay, mild intellectual disability, motor dysfunction, and progressive spasticity. Patient-derived fibroblasts, neural stem cells (NSCs), and in vivo zebrafish modeling were used to investigate pathomechanisms, including initial preclinical therapy assessment. The absence of AMFR disturbs lipid homeostasis, causing lipid droplet accumulation in NSCs and patient-derived fibroblasts which is rescued upon AMFR re-expression. Electron microscopy indicates ER morphology alterations in the absence of AMFR. Similar findings are seen in amfra-/- zebrafish larvae, in addition to altered touch-evoked escape response and defects in motor neuron branching, phenocopying the HSP observed in patients. Interestingly, administration of FDA-approved statins improves touch-evoked escape response and motor neuron branching defects in amfra-/- zebrafish larvae, suggesting potential therapeutic implications. Our genetic and functional studies identify bi-allelic truncating variants in AMFR as a cause of a novel autosomal recessive HSP by altering lipid metabolism, which may potentially be therapeutically modulated using precision medicine with statins.

In mammals, the transcription of transposable elements (TEs) is important for maintaining early embryonic development. Here, we systematically analyzed the expression characteristics of TE-derived transcripts in early embryos by constructing a database of TEs and transcriptome data from goats and using it to study the function of endogenous retroviruses (ERVs) in regulating early embryo development. We found that ERV1 made up the highest proportion of TE sequences and exhibited a stage-specific expression pattern during early embryonic development. Among ERV elements, ERV1 had the potential to encode the Gag protein domain to form virus-like particles (VLPs) in early goat embryos. Knockdown of ERV1_1_574 significantly reduced the embryo development rate and the number of trophoblast cells (P< 0.05). Transcriptome sequencing analysis of morula embryos showed that ERV1_1_574 mainly regulated the expression of genes related to embryo compaction and trophoblast cell differentiation, such as CX43 and CDX2. In summary, we found that ERV1 expression was essential for early embryonic development in goats through regulation of trophoblast cell differentiation.

Background Non-coding regulatory elements (NCREs), such as enhancers, play a crucial role in gene regulation, and genetic aberrations in NCREs can lead to human disease, including brain disorders. The human brain is a complex organ that is susceptible to numerous disorders; many of these are caused by genetic changes, but a multitude remain currently unexplained. Understanding NCREs acting during brain development has the potential to shed light on previously unrecognized genetic causes of human brain disease. Despite immense community-wide efforts to understand the role of the non-coding genome and NCREs, annotating functional NCREs remains challenging. Methods Here we performed an integrative computational analysis of virtually all currently available epigenome data sets related to human fetal brain. Results Our in-depth analysis unravels 39,709 differentially active enhancers (DAEs) that show dynamic epigenomic rearrangement during early stages of human brain development, indicating likely biological function. Many of these DAEs are linked to clinically relevant genes, and functional validation of selected DAEs in cell models and zebrafish confirms their role in gene regulation. Compared to enhancers without dynamic epigenomic rearrangement, DAEs are subjected to higher sequence constraints in humans, have distinct sequence characteristics and are bound by a distinct transcription factor landscape. DAEs are enriched for GWAS loci for brain-related traits and for genetic variation found in individuals with neurodevelopmental disorders, including autism. Conclusion This compendium of high-confidence enhancers will assist in deciphering the mechanism behind developmental genetics of human brain and will be relevant to uncover missing heritability in human genetic brain disorders.

Human post-natal neurodevelopmental delay is often associated with cerebral alterations that can lead, by themselves or associated with peripheral deficits, to premature death. Here, we report the clinical features of 10 patients from six independent families with mutations in the autosomal YIF1B gene encoding a ubiquitous protein involved in anterograde traffic from the endoplasmic reticulum to the cell membrane, and in Golgi apparatus morphology. The patients displayed global developmental delay, motor delay, visual deficits with brain MRI evidence of ventricle enlargement, myelination alterations and cerebellar atrophy. A similar profile was observed in the Yif1b knockout (KO) mouse model developed to identify the cellular alterations involved in the clinical defects. In the CNS, mice lacking Yif1b displayed neuronal reduction, altered myelination of the motor cortex, cerebellar atrophy, enlargement of the ventricles, and subcellular alterations of endoplasmic reticulum and Golgi apparatus compartments. Remarkably, although YIF1B was not detected in primary cilia, biallelic YIF1B mutations caused primary cilia abnormalities in skin fibroblasts from both patients and Yif1b-KO mice, and in ciliary architectural components in the Yif1b-KO brain. Consequently, our findings identify YIF1B as an essential gene in early post-natal development in human, and provide a new genetic target that should be tested in patients developing a neurodevelopmental delay during the first year of life. Thus, our work is the first description of a functional deficit linking Golgipathies and ciliopathies, diseases so far associated exclusively to mutations in genes coding for proteins expressed within the primary cilium or related ultrastructures. We therefore propose that these pathologies should be considered as belonging to a larger class of neurodevelopmental diseases depending on proteins involved in the trafficking of proteins towards specific cell membrane compartments.

Interstitial deletions on the long arm of chromosome 12 (12q deletions) are rare, and are associated with intellectual disability, developmental delay, failure to thrive and congenital anomalies. The precise genotype-phenotype correlations of different deletions has not been completely resolved. Ascertaining individuals with overlapping deletions and complex phenotypes may help to identify causative genes and improve understanding of 12q deletion syndromes. We here describe two individuals with non-overlapping 12q14 deletions encountered at our clinical genetics outpatient clinic and perform a review of all previously published interstitial 12q deletions to further delineate genotype-phenotype correlations. Both individuals presented with a neurodevelopmental disorder with various degrees of intellectual disability, failure to thrive and dysmorphic features. Previously, larger deletions overlapping large parts of the deletions encountered in both individuals have been described. Whereas, individual 1 seems to fit into the previously described phenotypic spectrum of the 12q14 microdeletion syndrome, individual 2 displays more severe neurological symptoms, which are likely caused by haploinsufficiency of the BAF complex member SMARCC2, which is included in the deletion. We furthermore perform a review of all previously published interstitial 12q deletions which we found to cluster amongst 5 regions on chromosome 12, to further delineate genotype-phenotype correlations, and we discuss likely disease relevant genes for each of these deletion clusters. Together, this expands knowledge on deletions on chromosome 12q which might facilitate patient counseling. Also, it illustrates that re-analysis of previously described microdeletions syndromes in the next generation sequencing era can be useful to delineate genotype-phenotype correlations and identify disease relevant genes in individuals with neurodevelopmental disorders.

Ten-eleven translocation 3 (TET3) mediates active DNA demethylation of paternal genomes during mouse embryonic development. However, the mechanism of DNA demethylation in goat embryos remains unknown. In addition, aberrant DNA methylation reprogramming prevalently occurs in embryos cloned by somatic cell nuclear transfer (SCNT). In this study, we reported that TET3 is a key factor in DNA demethylation in goat pre-implantation embryos. Knockdown of Tet3 hindered DNA demethylation at the two- to four-cell stage in goat embryos and decreased Nanog expression in blastocysts. Overexpression of Tet3 in somatic cells can initiate DNA demethylation, reduce 5-methylcytosine level, increase 5-hydroxymethylcytosine level and promote the expression of key pluripotency genes. After SCNT, overexpression of Tet3 in donor cells corrected abnormal DNA hypermethylation of cloned embryos and significantly enhanced in vitro and in vivo developmental rate (P < 0.05). We conclude that overexpression of Tet3 in donor cells significantly improves goat SCNT efficiency.

Epigenetic modifications extensively occur in mammalian embryonic development and cell differentiation process. They play an essential role in the reprogramming of nuclei during somatic cell nuclear transfer (SCNT) and subsequent in vitro embryonic development. Recently, SCNT embryos have been verified to contain a subnormal level of histone H3K4 dimethylation (H3K4me2) in contrast to in vitro fertilized embryos. This finding suggested that increasing H3K4me2 levels may ameliorate the aberrant development of cloned embryos. In this study, we investigated the influence of treating donor cells with trans-2-Phenylcyclopropylamine (2-PCPA), a specific inhibitor of lysine-specific demethylase 1 (LSD1), on embryogenesis, H3K4me2 level, and gene expression in cloned goat embryos. Treated goat fetal fibroblast cells (GFFs) with 2-PCPA served as donor cells for subsequent SCNT. Results showed that H3K4me2 levels in treated GFFs increased gradually with the increasing 2-PCPA concentration (p 0.05) and had no obvious influence in cell viability. The 2-PCPA-induced up-regulation of H3K4me2 levels led to G0/G1 cell cycle arrest and the difference was significant at 2μM compared with the control group (p 0.05). Interestingly, the development rate of goat SCNT embryos in vitro was significantly improved and aberrant H3K4me2 levels were effectively corrected in 2-PCPA-treated SCNT embryos in contrast to that in SCNT control embryos. Moreover, 2-PCPA treatment promoted the mRNA expression of key developmental genes Oct4 and Sox2 (p 0.05) without affecting the expression levels of imprinted genes IGF2R and H19 in goat SCNT embryos. These results indicated that abnormal H3K4me2 status can be corrected and SCNT embryo development can be promoted through treatment of donor cells with 2-PCPA.SCNT: somatic cell nuclear transfer; H3K4me2: H3K4 dimethylation; 2-PCPA: trans-2-Phenylcyclopropylamine; LSD1: lysine-specific demethylase 1; GFFs: goat fetal fibroblast cells; IVF: in vitro fertilization; iPS: induced pluripotent stem; PBS: phosphate-buffered saline; IVM: in vitro maturation; RNAPII: RNA polymerase II; HMTs: histone methyltransferase.

Quercetin, a plant-derived flavonoid, is frequently used as an antioxidant for efficient anti-oxidative capacity. However, whether quercetin has protective effects on goat sperm and preimplantation embryos against Cd2+-induced oxidative injury is still unclear. So, we researched the influence of quercetin on goat sperm and zygotes respectively under the oxidative stress induced by Cd2+. In our study, quercetin decreased the malonaldehyde (MDA) and reactive oxygen species (ROS) levels caused by Cd2+ in goat sperm (p

Epigenetic modifications are considered crucial to the reprogramming of somatic cell nuclear transfer (SCNT) embryos and subsequent in vitro development. The abnormal histone H3K9 dimethylation (H3K9me2) status has recently been reported in SCNT embryos. The present study was designed to evaluate the effect of treatment donor cells with UNC0638, a specific inhibitor of H3K9 methyltransferase G9A, on in vitro development, H3K9me2 levels and gene expression in goat SCNT embryos. Different concentration of UNC0638 was applied to treat goat fetal fibroblasts (GFFs), and the treated GFFs were used as donor cells for SCNT. The results showed that 0.2 μM UNC0638 decreased significantly the level of H3K9me2 in treated GFFs (P 0.05). Compared with intracytoplasmic sperm injection (ICSI) embryos, SCNT embryos derived from untreated GFFs (C-SCNT) presented higher level of H3K9me2 (P 0.05). In conclusion, these results indicated that treatment donor cells with UNC0638 may have beneficial effects in terms of modifying the abnormal H3K9me2 status and gene expression in cloned goat embryos.

Lipopolysaccharide (LPS) and lipoteichoic acid (LTA) are key virulence factors of Escherichia coli and Staphylococcus aureus respectively, and both of them could cause inflammatory reaction in bovine mammary glands. In this study, we used bovine mammary epithelial cells (BMECs) as pattern recognition receptors and stimulated them with LPS or LTA to investigate the global transcriptional response variations of BMECs to these two different virulent factors through RNA-Seq analysis. We found 100 differentially expressed genes (DEGs) with 95 up-regulated and 5 down-regulated genes in LPS-treated group, whereas 24 DEGs with 12 up-regulated and 12 down-regulated genes in LTA-treated group compared to control. Although the number and expression changes of DEGs are significantly different between LPS vs Control and LTA vs Control, KEGG pathway enrichment analysis showed the majorities of DEGs in each pair were enriched on cytokine-cytokine receptor interaction, NF-κB signaling pathway, and NOD-like receptor signaling pathway, especially cytokines and chemokines. These results provided a comprehensive analysis of gene expression profiles elicited by LPS and LTA in BMECs, contributing to the understanding of early "pathogen-host" interactions during intramammary infections.

Endogenous retroviruses (ERVs), which are abundant in mammalian genomes, can modulate the expression of nearby genes, and their expression is dynamic and stage-specific during early embryonic development in mice and humans. However, the functions and mechanisms of ERV elements in regulating embryonic development remain unclear. Here, we utilized several methods to determine the contribution of ERVs to the makeup and regulation of transcripts during embryonic genome activation (EGA). We constructed an ERV library and embryo RNA-seq library (IVF_2c and IVF_8c) of goat to serve as our research basis. The GO and KEGG analysis of nearby ERV genes revealed that some ERV elements may be associated with embryonic development. RNA-seq results were consistent with the features of EGA. To obtain the transcripts derived from the ERV sequences, we blasted the ERV sequences with embryonic transcripts and identified three lncRNAs and one mRNA that were highly expressed in IVF-8c rather than in IVF-2c (q-value

We explore a key architectural aspect of deep convolutional neural networks: the pattern of internal skip connections used to aggregate outputs of earlier layers for consumption by deeper layers. Such aggregation is critical to facilitate training of very deep networks in an end-to-end manner. This is a primary reason for the widespread adoption of residual networks, which aggregate outputs via cumulative summation. While subsequent works investigate alternative aggregation operations (e.g. concatenation), we focus on an orthogonal question: which outputs to aggregate at a particular point in the network. We propose a new internal connection structure which aggregates only a sparse set of previous outputs at any given depth. Our experiments demonstrate this simple design change offers superior performance with fewer parameters and lower computational requirements. Moreover, we show that sparse aggregation allows networks to scale more robustly to 1000+ layers, thereby opening future avenues for training long-running visual processes.

Deep Neural Networks (DNNs) have been widely applied in various recognition tasks. However, recently DNNs have been shown to be vulnerable against adversarial examples, which can mislead DNNs to make arbitrary incorrect predictions. While adversarial examples are well studied in classification tasks, other learning problems may have different properties. For instance, semantic segmentation requires additional components such as dilated convolutions and multiscale processing. In this paper, we aim to characterize adversarial examples based on spatial context information in semantic segmentation. We observe that spatial consistency information can be potentially leveraged to detect adversarial examples robustly even when a strong adaptive attacker has access to the model and detection strategies. We also show that adversarial examples based on attacks considered within the paper barely transfer among models, even though transferability is common in classification. Our observations shed new light on developing adversarial attacks and defenses to better understand the vulnerabilities of DNNs.