GIS-based intelligent planning approach of child-friendly pedestrian pathway to promote a child-friendly city.

Pedestrian safety, particularly for children, relies on well-designed pathways. Child-friendly pathways play a crucial role in safeguarding young pedestrians. Shared spaces accommodating both vehicles and walkers can bring benefits to pedestrians. However, active children playing near these pathways are prone to accidents. This research aims to develop an efficient method for planning child-friendly pedestrian pathways, taking into account community development and the specific needs of children. A mixed-methods approach was employed, utilizing the Datang community in Guangzhou, China, as a case study. This approach combined drawing techniques with GIS data analysis. Drawing methods were utilized to identify points of interest for children aged 2-6. The qualitative and quantitative fuzzy analytic hierarchy process assessed factors influencing pathway planning, assigning appropriate weights. The weighted superposition analysis method constructed a comprehensive cost grid, considering various community elements. To streamline the planning process, a GIS tool was developed based on the identified factors, resulting in a practical, child-friendly pedestrian pathway network. Results indicate that this method efficiently creates child-friendly pathways, ensuring optimal connectivity within the planned road network.

Research on student engagement in distance learning in sustainability science to design an online intelligent assessment system.

Distance learning programs in sustainability science provide a structured curriculum that covers various aspects of sustainability. Despite the growing recognition of distance learning in higher education, existing literature has primarily focused on specific and detailed factors, without a comprehensive summary of the global themes, especially neglecting in-depth exploration of poor engagement factors. This study bridged this gap by not only examining detailed factors but also synthesizing the overarching themes that influenced student engagement. The aim of this study was to investigate the factors that impact student engagement in distance learning within higher education institutions across different countries. By developing a theoretical framework, three key aspects of student engagement in higher education were identified. A total of 42 students and 2 educators affiliated with universities participated in semi-structured interviews. The findings of this paper indicated that sociocultural, infrastructure, and digital equity factors were the main influencing factors of student engagement. Furthermore, a student engagement assessment system was developed using machine learning algorithms to identify students with low levels of engagement and conduct further analysis that considers the three aforementioned factors. The proposed automated approach holds the potential to enhance and revolutionize digital learning methodologies.

Influence of dynamic load and water on energy accumulation and dissipation in sandstone.

In various engineering projects such as mineral extraction, hydropower resource utilization, railway construction, and geological hazard mitigation, rock engineering is often encountered. Furthermore, dynamic loads and moisture content exert notable influence on the energy transformation processes within rocks. Yet, the specific interplay of dynamic loading and water's impact on the energy conversion mechanism within the sandstone remains unexplored. To address this gap, this study conducted impact loading experiments on sandstone, elucidating the rock's mechanical response under these conditions and unraveling the underlying energy conversion mechanisms. It was observed that the strength of sandstone exhibits a direct correlation with impact velocity. Moreover, employing energy calculation principles, we established a connection between moisture content and the sandstone's internal energy conversion properties. The study also delved into the microscopic fracture mechanisms within the sandstone, ultimately concluding that both water content and dynamic loading have a significant impact on these microscopic fracture mechanisms.

ZFP541 and KCTD19 regulate chromatin organization and transcription programs for male meiotic progression.

The successful progression of meiosis prophase I requires integrating information from the structural and molecular levels. In this study, we show that ZFP541 and KCTD19 work in the same genetic pathway to regulate the progression of male meiosis and thus fertility. The Zfp541 and/or Kctd19 knockout male mice show various structural and recombination defects including detached chromosome ends, aberrant localization of chromosome axis components and recombination proteins, and globally altered histone modifications. Further analyses on RNA-seq, ChIP-seq, and ATAC-seq data provide molecular evidence for the above defects and reveal that ZFP541/KCTD19 activates the expression of many genes by repressing several major transcription repressors. More importantly, we reveal an unexpected role of ZFP541/KCTD19 in directly modulating chromatin organization. These results suggest that ZFP541/KCTD19 simultaneously regulates the transcription cascade and chromatin organization to ensure the coordinated progression of multiple events at chromosome structural and biochemical levels during meiosis prophase I.

TTC6-Mediated Stabilization of the Flagellum Annulus Ensures the Rapid and Directed Motion of Sperm.

Sperm motility and structural integrity are essential for successful fertilization in vivo, and any hindrance of the correct assembly of the axoneme and peri-axonemal structures in the sperm flagellum can lead to fertility problems. While there has been considerable advancement in studying diseases related to the flagellum, the underlying mechanisms that control sperm movement are not yet fully understood. In this study, we reveal that the tetratricopeptide repeat protein 6 (Ttc6) gene, expressed mainly in the testes, plays a crucial role in maintaining male fertility in mice. We further demonstrate that the knockout of Ttc6 in mice results in decreased sperm motility and induces an abnormal circular swimming pattern, consequently leading to male subfertility. Morphological analysis showed an atypical hairpin-like appearance of the spermatozoa, and ultrastructural studies showed unsheathed flagella at the juncture between the midpiece and principal piece. Collectively, these findings suggest that TTC6 plays an essential role in maintaining the stability of the annulus region of the sperm flagellum, thus ensuring the swift and directed motion of sperm.

The Slingshot phosphatase 2 is required for acrosome biogenesis during spermatogenesis in mice.

The acrosome is a membranous organelle positioned in the anterior portion of the sperm head and is essential for male fertility. Acrosome biogenesis requires the dynamic cytoskeletal shuttling of vesicles toward nascent acrosome which is regulated by a series of accessory proteins. However, much remains unknown about the molecular basis underlying this process. Here, we generated Ssh2 knockout (KO) mice and HA-tagged Ssh2 knock-in (KI) mice to define the functions of Slingshot phosphatase 2 (SSH2) in spermatogenesis and demonstrated that as a regulator of actin remodeling, SSH2 is essential for acrosome biogenesis and male fertility. In Ssh2 KO males, spermatogenesis was arrested at the early spermatid stage with increased apoptotic index and the impaired acrosome biogenesis was characterized by defective transport/fusion of proacrosomal vesicles. Moreover, disorganized F-actin structures accompanied by excessive phosphorylation of COFILIN were observed in the testes of Ssh2 KO mice. Collectively, our data reveal a modulatory role for SSH2 in acrosome biogenesis through COFILIN-mediated actin remodeling and the indispensability of this phosphatase in male fertility in mice.

Ptbp1 knockdown failed to induce astrocytes to neurons in vivo.

The conversion of non-neuronal cells to neurons is a promising potential strategy for the treatment of neurodegenerative diseases. Recent studies have reported that shRNA-, CasRx-, or ASO-mediated Ptbp1 suppression could reprogram resident astrocytes to neurons. However, some groups have disputed the interpretation of the data underlying the reported neuron conversion events. These controversies surrounding neuron conversion may be due to differences in the astrocyte fate-mapping systems. Here, we suppressed Ptbp1 using Cas13X and labelled astrocytes with an HA tag fused to Cas13X (Cas13X-NLS-HA). We found no astrocyte-to-neuron conversion in the mouse striatum via the HA-tagged labelling system compared with the GFAP-driven tdTomato labelling system (AAV-GFAP::tdTomato-WPRE) used in previous studies. Our findings indicate that Cas13X-mediated Ptbp1 knockdown failed to induce neuron conversion in vivo.

High-fidelity Cas13 variants for targeted RNA degradation with minimal collateral effects.

CRISPR-Cas13 systems have recently been used for targeted RNA degradation in various organisms. However, collateral degradation of bystander RNAs has limited their in vivo applications. Here, we design a dual-fluorescence reporter system for detecting collateral effects and screening Cas13 variants in mammalian cells. Among over 200 engineered variants, several Cas13 variants including Cas13d and Cas13X exhibit efficient on-target activity but markedly reduced collateral activity. Furthermore, transcriptome-wide off-targets and cell growth arrest induced by Cas13 are absent for these variants. High-fidelity Cas13 variants show similar RNA knockdown activity to wild-type Cas13 but no detectable collateral damage in transgenic mice or adeno-associated-virus-mediated somatic cell targeting. Thus, high-fidelity Cas13 variants with minimal collateral effects are now available for targeted degradation of RNAs in basic research and therapeutic applications.

Safeguarding genome integrity during gene-editing therapy in a mouse model of age-related macular degeneration.

Ensuring genome safety during gene editing is crucial for clinical translation of the high-efficient CRISPR-Cas9 toolbox. Therefore, the undesired events including chromosomal translocations, vector integrations, and large deletions arising during therapeutic gene editing remain to be adequately addressed or tackled in vivo. Here, we apply CRISPR-Cas9TX in comparison to CRISPR-Cas9 to target Vegfa for the treatment of age-related macular degeneration (AMD) disease in a mouse model. AAV delivery of both CRISPR-Cas9 and CRISPR-Cas9TX can efficiently inhibit laser-induced neovascularization. Importantly, Cas9TX almost eliminates chromosomal translocations that occur at a frequency of approximately 1% in Cas9-edited mouse retinal cells. Strikingly, the widely observed AAV integration at the target Vegfa site is also greatly reduced from nearly 50% of edited events to the background level during Cas9TX editing. Our findings reveal that chromosomal structural variations routinely occur during in vivo genome editing and highlight Cas9TX as a superior form of Cas9 for in vivo gene disruption.

Develop an efficient and specific AAV-based labeling system for Muller glia in mice.

Reprogramming Müller glia (MG) into functional cells is considered a promising therapeutic strategy to treat ocular diseases and vision loss. However, current AAV-based system for MG-tracing was reported to have high leakage in recent studies. Here, we focused on reducing the leakage of AAV-based labeling systems and found that different AAV serotypes showed a range of efficiency and specificity in labeling MG, leading us to optimize a human GFAP-Cre reporter system packaged in the AAV9 serotype with the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) removed. The leakage ratio of the AAV9-hGFAP-Cre-ΔWPRE decreased by an approximate 40-fold compared with the AAV9-hGFAP-Cre-WPRE labeling system. In addition, we validated the specificity of the AAV-ΔWPRE system for tracing MG reprogramming under Ptbp1-suppression and observed strict non-MG-conversion, similar to previous studies using genetic lineage tracking mouse models. Thus, the AAV9-hGFAP-Cre-ΔWPRE system showed high efficiency and specificity for MG labeling, providing a promising tool for tracing cell fate in vivo.

Dynamic Responses of Chromosome-Binding Protein Complexes to Meiotic Prophase I of Mouse Spermatocyte.

Meiotic prophase I (MPI) is the most important event in mammalian meiosis. The status of the chromosome-binding proteins (CBPs) and the corresponding complexes and their functions in MPI have not yet been well scrutinized. Quantitative proteomics focused on MPI-related CBPs was accomplished, in which mouse primary spermatocytes in four different subphases of MPI were collected, and chromosome-enriched proteins were extracted and quantitatively identified. According to a stringent criterion, 1136 CBPs in the MPI subphases were quantified. Looking at the dynamic patterns of CBP abundance in response to MPI progression, the patterns were broadly divided into two groups: high abundance in leptotene and zygotene or that in pachytene and diplotene. Furthermore, 152 such CBPs were regarded as 26 CBP complexes with strict filtration, in which some of these complexes were perceived to be MPI-dependent for the first time. These complexes basically belonged to four functional categories, while their dynamic abundance changes following MPI appeared; the functions of DNA replication decreased; and transcription and synapsis were activated in zygotene, pachytene, and diplotene; in contrast to the traditional prediction, condensin activity weakened in pachytene and diplotene. Profiling of protein complexes thus offered convincing evidence of the importance of CBP complexes in MPI.

Prediction and Validation of Mouse Meiosis-Essential Genes Based on Spermatogenesis Proteome Dynamics.

The molecular mechanism associated with mammalian meiosis has yet to be fully explored, and one of the main reasons for this lack of exploration is that some meiosis-essential genes are still unknown. The profiling of gene expression during spermatogenesis has been performed in previous studies, yet few studies have aimed to find new functional genes. Since there is a huge gap between the number of genes that are able to be quantified and the number of genes that can be characterized by phenotype screening in one assay, an efficient method to rank quantified genes according to phenotypic relevance is of great importance. We proposed to rank genes by the probability of their function in mammalian meiosis based on global protein abundance using machine learning. Here, nine types of germ cells focusing on continual substages of meiosis prophase I were isolated, and the corresponding proteomes were quantified by high-resolution MS. By combining meiotic labels annotated from the mouse genomics informatics mouse knockout database and the spermatogenesis proteomics dataset, a supervised machine learning package, FuncProFinder (https://github.com/sjq111/FuncProFinder), was developed to rank meiosis-essential candidates. Of the candidates whose functions were unannotated, four of 10 genes with the top prediction scores, Zcwpw1, Tesmin, 1700102P08Rik, and Kctd19, were validated as meiosis-essential genes by knockout mouse models. Therefore, mammalian meiosis-essential genes could be efficiently predicted based on the protein abundance dataset, which provides a paradigm for other functional gene mining from a related abundance dataset.