Quantifying Desiccation Cracks for Expansive Soil Using Machine Learning Technique in Image Processing

The formation of desiccation cracks has detrimental effects on the hydraulic conductivity that affects the overall mechanical strength of expansive soil. Qualitative analysis on the desiccation cracking behaviour of expansive soil provided understanding of the subject based on various concepts and theories, while quantitative analysis aided these studies through numerical supports. In this study, a machine learning technique in image processing is developed to evaluate the surface crack ratio of expansive soil. The desiccation cracking tests were conducted on highly plastic kaolinite slurry samples with plasticity index of 29.1%. Slurry-saturated specimens with thickness of 10 mm were prepared. The specimens were subjected to cyclic drying-wetting conditions. The images are acquired through a digital camera (12 MP) at constant distance to monitor the desiccation cracks. The images are then pre-processed using OpenCV before crack feature extraction. In this study, a total of 54 desiccation crack images were processed, along with 8 images from trial test to train the model. The processed images are used to quantify the desiccation cracks by evaluating surface crack ratio and average crack width. It was identified that the accuracy of the model for the quantification of surface crack ratio and average crack width were 97.24% and 93.85% respectively with average processing time of 1.51s per image. The results show that the model was able to achieve high accuracy with sufficient efficiency in determining important parameters used for crack characterization

The role of JAK-STAT signalling in the developing brain of Ts1Cje mouse model for Down syndrome

Introduction: The JAK-STAT signalling pathway is essential for proper regulation of the gliogenesis process during brain development. Both Down syndrome (DS) individuals and DS mouse model showed reduced number of neuron but increase number of astrocyte in the brain; suggesting that dysregulation of JAK-STAT signalling pathway may have occurred and led to neurogenic-to-gliogenic shift in the DS brain. Such a shift in the DS brain may contribute to the intellectual disability seen in the DS individual and learning and memory impairment in the mouse model. Therefore, understanding the role JAK-STAT signalling in the DS brain at early stage may implicate the underlying mechanism(s) causing the shift and provide novel therapeutics targets for cognition therapy among DS individuals. Method: In this study, the whole brain of Ts1Cje and disomic mice was collected at embryonic day (E) 10.5, E14 and postnatal day (P) 1.5. Here, we focus on Jak1, Jak2, Stat1, Stat3 and Stat6, which have been shown to express highly or stably during early brain development. Results: Both RT-qPCR and western blot analysis revealed the expression of Jak1 as significantly reduced in Ts1Cje at E14. In addition, phosphorylated Jak2 and Stat6 expression levels were decreased in Ts1Cje at E14 as compared to disomic mice. Our findings suggest that JAK1 is important for astrocytic differentiation while JAK2 is essential for neural stem cells proliferation and STAT6 is crucial in mediating immune signalling. Thus, further investigation on the function of Jak1, Jak2 and Stat6 may provide insight into neurogenic-to-gliogenic shift occur in the DS brain

The expression profile of miR-3099 during neural development of Ts1Cje mouse model of down syndrome

MicroRNA-3099 (miR-3099) plays a crucial role in regulating neuronal differentiation and development of the central nervous system (CNS). The miR-3099 is a pro-neuronal miRNA that promotes neural stem/progenitor cell (NSPC) differentiation into neuronal lineage by suppressing astrogliogenesis. Down syndrome (DS) brain exhibited increased astrogliogenesis and reduced neuronal cell density. The involvement of miR-3099 in the neurodevelopment of DS has not been investigated and potentially responsible for the neurogenic-to-gliogenic shift phenomenon observed in DS brain. To investigate the role of miR-3099 during DS brain development, neural/progenitor cell proliferation and differentiation, we profiled miR-3099 expression level in the Ts1Cje, a mouse model for DS. We analysed the Ts1Cje whole brain at embryonic day (E) 10.5, E14.5 and P1.5, proliferating neurospheres and differentiating neurospheres at 3, 9 and 15 days in vitro (DIV). Expression of miR-3099 in both the developing mouse brain and the differentiating neurosphere was not significantly different between Ts1Cje and wild type controls. In contrast, the expression level of miR-3099 was significantly higher (p<0.05) in proliferating NSPC derived from the Ts1Cje compared to wild-type. Further molecular profiling of NPSC and glial cell markers indicated that the expression of Sox2 (p<0.01) and Gfap (p<0.05) were significantly downregulated in Ts1Cje neurospheres as compared to that of wild type, respectively. While there were no significant differences in Tuj1 and Nestin expression levels between the Ts1Cje and wild type neurospheres, their expression levels were ~3-fold upregulated and ~2.6 downregulated Ts1Cje group, respectively. The findings suggest that dysregulation of miR-3099 affects NSPC lineage commitment as indicated by altered postmitotic neuronal cell markers. Further molecular characterisation and gene expression profiling of other neuronal and glial markers will help refine the analysis of gene-gene interactions underlying the neuropathologies of DS

Construction and validation of a mammalian expression vector for in utero electroporation study of miR-3099 in the mouse neocortex

Introduction: MiR-3099 was reported to play a role in neuronal cell differentiation/function in the brain during late embryonic and early neonatal development. To further explore its potential regulatory effects on embryonic brain development, this study aims to construct and validate an expression vector of miR-3099 for future gain-of-function and loss-of-function studies. Methods: pCAG-eGFP vector was modified to include IRES2 and miR-3099 with 150bp upstream and downstream genomic sequences. The newly constructed vector, pCAG-miR-3099-IRES2-eGFP, consists of CAG promoter. The in vitro expression level of miR-3099 was measured using stem-loop RT-qPCR after it was transfected into 293FT cell. Later, the vector was electroporated into the embryonic brain at E15.5. Three days later, the E18.5 embryonic brain was harvested and cryopreserved. Immunohistochemistry was performed by using antibody against eGFP to validate the in utero expression of the transgene in the neocortex of the brain. Results: Our finding showed that, the expression level of miR-3099 was significantly upregulated (p<0.001) in cells transfected with miR-3099 vector as compared to both negative and empty plasmid control groups. In addition, the expression of eGFP was noted in the brain section indicating that the vectors with or without miR-3099 transgene were successfully transfected into and expressed in the neocortex upon electroporation. Conclusion: The bicistronic expression vector of miR-3099 which was driven by the CAG promoter was successfully constructed, validated and sufficiently delivered to brain cells via the in utero electroporation approach. The regulatory roles of miR-3099 in embryonic brain development can be manipulated using similar approach

Disrupted interferon-related molecular networks and the over-expressed Ifnar1 in the brain of adult Ts1Cje mouse model of Down syndrome

Down syndrome (DS) is a chromosomal disorder resulting from trisomy of human chromosome 21 (HSA21) and all DS individual exhibit cognitive impairment. Ts1Cje mouse model of DS has a triplicated region of mouse chromosome 16 (MMU16) which is homologous to HSA21. Three interferon receptor genes (Ifnar1, Ifnar2 and Ifngr2) are located at the triplicated region in MMU16 and also in HSA21. In this study, we aimed to determine the disrupted molecular networks and the role of the candidate gene in the neurogenic-to-gliogenic shift of Ts1Cje mouse brain. A functional transcriptome analysis was performed on the cerebral cortex, cerebellum and hippocampus of Ts1Cje mice at 4 time-points: postnatal day (P)1, P15, P30 and P84. Functional clustering analysis of the identified 317 differentially expressed genes reported interferon-related signalling networks as the most significantly dysregulated pathway in Ts1Cje postnatal brain. Both Ifnar1 and Stat1 were found over-expressed in P84 Ts1Cje cerebral cortex and cerebellum when compared to wild type littermates through qRT-PCR and western blotting analysis. Subsequently, the role of triplicated Ifnar1 was determined by treating Ifnar1 antagonist on differentiating neural stem cells derived from the SVZ of adult Ts1Cje. The assessment on the antagonistic effect of Ifnar1 antagonist reported successful attenuation on the aberrant Stat1 expression in the Ts1Cje group to an expression level which was similar to the wild type group

Derivation of an endogenous small RNA from double-stranded Sox4 sense and natural antisense transcripts in the mouse brain

Natural antisense transcripts (NATs) are involved in cellular development and regulatory processes. Multiple NATs at the Sox4 gene locus are spatiotemporally regulated throughout murine cerebral corticogenesis. In the study, we evaluated the potential functional role of Sox4 NATs at Sox4 gene locus. We demonstrated Sox4 sense and NATs formed dsRNA aggregates in the cytoplasm of brain cells. Over expression of Sox4 NATs in NIH/3T3 cells generally did not alter the level of Sox4 mRNA expression or protein translation. Upregulation of a Sox4 NAT known as Sox4ot1 led to the production of a novel small RNA, Sox4_sir3. Its biogenesis is Dicer1-dependent and has characteristics resemble piRNA. Expression of Sox4_sir3 was observed in the marginal and germinative zones of the developing and postnatal brains suggesting a potential role in regulating neurogenesis. We proposed that Sox4 sense-NATs serve as Dicer1-dependent templates to produce a novel endo-siRNA- or piRNA-like Sox4_sir3

Overexpression of interferon alpha or beta receptors in the brain of adult Ts1Cje mouse model of Down syndrome

Introduction: Down syndrome (DS) is a generic disorder with trisomy of human chromosome 21 (HSA21) and all DS patients exhibited intellectual disability. Ts1Cje mouse model of DS has partial triplication of mouse chromosome 16 (MMU16) which is homologous to HSA21. The JAK (Janus kinase) and STAT (signal transducer and activator of transcription) signalling pathway is involved in neurogenesis and gliogenesis regulation. Cytokines especially the interferons (IFN) family is the major activator of JAK-STAT signalling pathway. Furthermore, interferon receptor genes (Ifnar), Ifnar2 and Ifngr2 are located at the triplicated region in MMU16 and also in HSA21. Method: Gene expression of Ifnar1, Ifnar2, Ifngr2 and associated genes in JAK-STAT signalling pathway (Jak1, Jak2, Stat1, Stat3 and Stat6) in the cerebral cortex and cerebellum between Ts1Cje and wild type control at four time-points; post natal day (P)1, P15, P30 and P84 was investigated by using qRT-PCR techniques. Western blotting was used to confirm the overexpression of Ifnar1, Ifnar2 and Stat1 in the cerebral cortex and cerebellum of Ts1Cje aged P84. Results: Ifnar1, Ifnar2, Ifngr2 and Stat1 were significantly overexpression in the cerebral cortex and cerebellum of Ts1Cje at various time points as compared to control littermates. Protein expression analysis confirmed the overexpression of Ifnar1 and Stat1 in the cerebellum of Ts1Cje mouse at P84 as compared to wild type. The findings suggest that overexpression of interferon receptors will increase sensitivity towards interferon levels in Ts1Cje mouse brain. Consequently, the over-stimulated JAK-STAT signalling pathway may contribute to the defective neurogenesis the Down syndrome mouse brain

In depth analysis of the Sox4 gene locus that consists of sense and natural antisense transcripts

SRY (Sex Determining Region Y)-Box 4 or Sox4 is an important regulator of the pan-neuronal gene expression during post-mitotic cell differentiation within the mammalian brain. Sox4 gene locus has been previously characterized with multiple sense and overlapping natural antisense transcripts [1] and [2]. Here we provide accompanying data on various analyses performed and described in Ling et al. [2]. The data include a detail description of various features found at Sox4 gene locus, additional experimental data derived from RNA-Fluorescence in situ Hybridization (RNA-FISH), Western blotting, strand-specific reverse-transcription quantitative polymerase chain reaction (RT-qPCR), gain-of-function and in situ hybridization (ISH) experiments. All the additional data provided here support the existence of an endogenous small interfering- or PIWI interacting-like small RNA known as Sox4_sir3, which origin was found within the overlapping region consisting of a sense and a natural antisense transcript known as Sox4ot1