Mechanical Characterization of Microengineered Epithelial Cysts by Using Atomic Force Microscopy.

Most organs contain interconnected tubular tissues that are one-cell-thick, polarized epithelial monolayers enclosing a fluid-filled lumen. Such tissue organization plays crucial roles in developmental and normal physiology, and the proper functioning of these tissues depends on their regulation by complex biochemical perturbations and equally important, but poorly understood, mechanical perturbations. In this study, by combining micropatterning techniques and atomic force microscopy, we developed a simple in vitro experimental platform for characterizing the mechanical properties of the MDCK II cyst, the simplest model of lumen-enclosing epithelial monolayers. By using this platform, we estimated the elasticity of the cyst monolayer and showed that the presence of a luminal space influences cyst mechanics substantially, which could be attributed to polarization and tissue-level coordination. More interestingly, the results from force-relaxation experiments showed that the cysts also displayed tissue-level poroelastic characteristics that differed slightly from those of single cells. Our study provides the first quantitative findings, to our knowledge, on the tissue-level mechanics of well-polarized epithelial cysts and offers new insights into the interplay between cyst mechanics and cyst physiology. Moreover, our simple platform is a potentially useful tool for enhancing the current understanding of cyst mechanics in health and disease.

Meta-analyses of methylation markers for prostate cancer.

Prostate cancer (PCa) is the most frequently diagnosed non-cutaneous cancer that has become the sixth leading cause of mortality in both the developed and developing countries. Accumulating evidence showed a number of genes with aberrant DNA methylation in the pathogenesis of PCa. Here, we conducted a systematic meta-analysis to evaluate the contribution of aberrantly methylated genes to the risk of PCa. Relevant methylation studies were retrieved from PubMed and Wanfang literature databases. In the meta-analysis, Mantel-Haenszel odds ratios (ORs) and 95 % confidence intervals (CIs) were calculated for each methylation event under appropriate models. A total of 594 publications were initially retrieved from PubMed and Wanfang literature database. After a three-step filtration, we harvested 39 case-control articles investigating the role of gene methylation in the prediction of PCa risk. Among the 31 genes involved, 24 genes were shown to be significantly hypermethylated in the PCa patients. Our meta-analyses identified strong associations of four aberrantly methylated genes (GSTP1, RASSF1, p16, and RARB) with PCa. Further research is needed to strengthen our findings in the future.

Microtubule-associated protein MAP7 promotes tubulin posttranslational modifications and cargo transport to enable osmotic adaptation.

Cells remodel their cytoskeletal networks to adapt to their environment. Here, we analyze the mechanisms utilized by the cell to tailor its microtubule landscape in response to changes in osmolarity that alter macromolecular crowding. By integrating live-cell imaging, ex vivo enzymatic assays, and in vitro reconstitution, we probe the impact of cytoplasmic density on microtubule-associated proteins (MAPs) and tubulin posttranslational modifications (PTMs). We find that human epithelial cells respond to fluctuations in cytoplasmic density by modulating microtubule acetylation, detyrosination, or MAP7 association without differentially affecting polyglutamylation, tyrosination, or MAP4 association. These MAP-PTM combinations alter intracellular cargo transport, enabling the cell to respond to osmotic challenges. We further dissect the molecular mechanisms governing tubulin PTM specification and find that MAP7 promotes acetylation and inhibits detyrosination. Our data identify MAP7 in modulating the tubulin code, resulting in microtubule cytoskeleton remodeling and alteration of intracellular transport as an integrated mechanism of cellular adaptation.

Macromolecular Crowding Tailors the Microtubule Cytoskeleton Through Tubulin Modifications and Microtubule-Associated Proteins.

Cells remodel their cytoskeletal networks to adapt to their environment. Here, we analyze the mechanisms utilized by the cell to tailor its microtubule landscape in response to changes in osmolarity that alter macromolecular crowding. By integrating live cell imaging, ex vivo enzymatic assays, and in vitro reconstitution, we probe the impact of acute perturbations in cytoplasmic density on microtubule-associated proteins (MAPs) and tubulin posttranslational modifications (PTMs), unraveling the molecular underpinnings of cellular adaptation via the microtubule cytoskeleton. We find that cells respond to fluctuations in cytoplasmic density by modulating microtubule acetylation, detyrosination, or MAP7 association, without differentially affecting polyglutamylation, tyrosination, or MAP4 association. These MAP-PTM combinations alter intracellular cargo transport, enabling the cell to respond to osmotic challenges. We further dissect the molecular mechanisms governing tubulin PTM specification, and find that MAP7 promotes acetylation by biasing the conformation of the microtubule lattice, and directly inhibits detyrosination. Acetylation and detyrosination can therefore be decoupled and utilized for distinct cellular purposes. Our data reveal that the MAP code dictates the tubulin code, resulting in remodeling of the microtubule cytoskeleton and alteration of intracellular transport as an integrated mechanism of cellular adaptation.

Global Trends in Green Space and Senior Mental Health Studies: Bibliometric Review.

The Sustainable Development Goals and the World Health Organization have prioritized senior mental health as an important goal. Senior mental health is a critical issue within the global public health sphere. Notably, green spaces are a useful alternative for improving senior mental health. Many studies have focused on green space and senior mental health, especially on their connection and relationship. However, this research topic lacks a comprehensive and systematic review. Owing to the lack of critical reviews, this study clarified the trend, progress, status, and focus of studies on green spaces and senior mental health using bibliometric analysis of literature within the Web of Science database. The literature analysis within this study specifically focused on the following, including the country/region contribution analysis, institution contribution analysis, keyword analysis, and highly productive journal analysis. Furthermore, this study systematically recorded the content of green space and senior mental health, identified the gap that exists, and provided future frontier directions or issues for research. These contribute toward comprehending the progress and content of this research topic and further provide a guide, reference, and inspiration for possible future research.

Nanobead-based single-molecule pulldown for single cells.

Investigation of cell-to-cell variability holds critical physiological and clinical implications. Thus, numerous new techniques have been developed for studying cell-to-cell variability, and these single-cell techniques can also be used to investigate rare cells. Moreover, for studying protein-protein interactions (PPIs) in single cells, several techniques have been developed based on the principle of the single-molecule pulldown (SiMPull) assay. However, the applicability of these single-cell SiMPull (sc-SiMPull) techniques is limited because of their high technical barrier and special requirements for target cells and molecules. Here, we report a highly innovative nanobead-based approach for sc-SiMPull that is based on our recently developed microbead-based, improved version of SiMPull for cell populations. In our sc-SiMPull method, single cells are captured in microwells and lysed in situ, after which commercially available, pre-surface-functionalized magnetic nanobeads are placed in the microwells to specifically capture proteins of interest together with their binding partners from cell extracts; subsequently, the PPIs are examined under a microscope at the single-molecule level. Relative to previously published methods, nanobead-based sc-SiMPull is considerably faster, easier to use, more reproducible, and more versatile for distinct cell types and protein molecules, and yet provides similar sensitivity and signal-to-background ratio. These crucial features should enable universal application of our method to the study of PPIs in single cells.

Single-frame deep-learning super-resolution microscopy for intracellular dynamics imaging.

Single-molecule localization microscopy (SMLM) can be used to resolve subcellular structures and achieve a tenfold improvement in spatial resolution compared to that obtained by conventional fluorescence microscopy. However, the separation of single-molecule fluorescence events that requires thousands of frames dramatically increases the image acquisition time and phototoxicity, impeding the observation of instantaneous intracellular dynamics. Here we develop a deep-learning based single-frame super-resolution microscopy (SFSRM) method which utilizes a subpixel edge map and a multicomponent optimization strategy to guide the neural network to reconstruct a super-resolution image from a single frame of a diffraction-limited image. Under a tolerable signal density and an affordable signal-to-noise ratio, SFSRM enables high-fidelity live-cell imaging with spatiotemporal resolutions of 30 nm and 10 ms, allowing for prolonged monitoring of subcellular dynamics such as interplays between mitochondria and endoplasmic reticulum, the vesicle transport along microtubules, and the endosome fusion and fission. Moreover, its adaptability to different microscopes and spectra makes it a useful tool for various imaging systems.

The stability and immunogenicity of formalin-inactivated Enterovirus A71 whole virion vaccine after ten years of low temperature storage.

BACKGROUND: Vaccine stability is an important issue for vaccine development, which affects whether the vaccine product is effective within a certain period of time in each progress. Hand, foot, and mouth diseases (HFMD) is an epidemic disease in young children usually caused by Enterovirus A group viruses, and the Enterovirus A71 (EV-A71) had caused several pandemics and public health issues around the world. After two decades of research and development, formalin-inactivated EV-A71 (FI-EV-A71) vaccines are the first to complete the phase III clinical trials for protection against EV-A71 infection. Currently, the shelf life of FI-EV-A71 vaccine product is set to be within 18 months, but the stability and the effectiveness of the FI-EV-A71 whole virion when stored long-term at low temperature remains undetermined. METHODS: Assessing the long-term storage properties of viral particles facilitates flexibility in manufacturing of vaccine products. In this study, the stability profiles of FI-EV-A71 vaccine lots and bulks after long-term of low temperature storage were analyzed by protein tests, particle measurement and animal immunization study. RESULTS: After over ten years of storage, the reduction of protein concentration in the FI-EV-A71 bulk samples is less than 30 % and the antigenic content remained in a suspended, particulate state. Both the packed FI-EV-A71 final vaccine products and the FI-EV-A71 antigens adjuvant premix bulk could elicit strong neutralizing responses in mice. CONCLUSION: After ten years of low temperature storage, the FI-EV-A71 vaccine still presents decent stability and good immunogenicity.

Propagation and immunological characterization of coxsackievirus A10 in a serum-free HEK293A cell culture system.

Coxsackievirus A10 (CVA10) is one of enteroviral pathogens that cause the hand, foot, and mouth disease (HFMD). Since CVA10 was reported to be not easily propagated in the Vero cell culture, a feasible manufacture process for producing formalin-inactivated CVA10 vaccine is urgently needed. Several cell lines that commonly used for viral vaccine production was tested for CVA10 (M2014 strain) culture in this study, and our result showed that CVA10 could be easily propagated in the HEK293A cells. A serum-free HEK293A cell culture system was developed for CVA10 production and the yields have reached over 108 TCID50/mL. The biochemical and immunogenic properties of CVA10 particles obtained from this serum-free HEK293A culture were identical to our previous study. Two major particles of CVA10 were separated by ultracentrifugation, and only the infectious mature particles were capable of inducing CVA10 neutralizing antibody responses in the mouse immunogenicity studies. Additionally, we found that coxsackievirus A6 and enterovirus A71 could also be easily propagated using this serum-free HEK293A cell culture system. Our results provide a solution to overcome the obstacle in the propagation of CVA10 and facilitate the development of multivalent vaccines for prevention of HFMD.

Multiple pathways and mediation effects of built environment on kidney disease rate via mitigation of atmospheric threats.

Air pollution and high temperatures can increase kidney disease rate, especially under climate change. A well-designed urban environment has mediating effects on atmospheric environmental threats and promoting human health, but previous studies have overlooked these effects. This study used partial least squares modeling and urban-scale data from Taiwan to identify the crucial effects (i.e., direct, indirect, and total effects) and pathways of urban form (i.e., urban development intensity, land-use mix, and urban sprawl), urban greening (i.e., green coverage), urban industrial status (e.g., industrial level), atmospheric environment (i.e., high temperature and air pollution), and socioeconomic status (i.e., elderly ratio, medical resources, and economic status) on kidney disease rate. Maximizing land-use mix and green coverage and minimizing urban development intensity, urban sprawl, and industrial levels could help reduce kidney disease rate. Air pollution and high temperature had a mediation effect of built environment on kidney disease rate; with the mediation effect of air pollution was greater than that of high temperature. Furthermore, air pollution, high temperature, and elderly ratio increased kidney disease rate, whereas medical resources decreased kidney disease rate. This study is the first to consider the impact (i.e., direct, indirect, and total effects) and pathways of built environment characteristics on kidney disease rate. The findings revealed that an appropriate urban policy might be a practical strategy and lower kidney disease rate for a healthy city development. Moreover, this study provides a new approach for clarifying complex relationships and identifying crucial factors.

Exploring multiple pathways and mediation effects of urban environmental factors for suicide prevention.

Public health is threatened by air pollution and high temperature, especially in urban areas and areas impacted by climate change. Well-designed urban forms have co-benefits on promoting human health and mediating atmospheric environment-related threats (e.g., high temperature and air pollution). Previous studies overlooked these mediating effects of urban form on suicide mortality. This study used partial least squares modeling and countywide data in Taiwan to identify the crucial influences and pathways of urban environment, socioeconomic status, and diseases on suicide mortality. The model considered the impact of the characteristics of urban form (i.e., urban development intensity, land mix, and urban sprawl), urban industrial status (i.e., industrial level), urban greening (i.e., green coverage), disease (i.e., important diseases morbidity of human immunodeficiency virus [HIV], cerebrovascular disease [CVD], chronic liver disease and cirrhosis [CLDC], nephritis, nephrotic syndrome and nephrosis [NNSN], malignant tumor [MT]), socioeconomic status (i.e., income level and aging population rate), and the atmospheric environment (i.e., air pollution and high temperature) on suicide mortality. Optimizing land mix and minimizing urban development intensity and urban sprawl have been found to reduce suicide mortality. The mediating effect of urban form on suicide mortality originated from air pollution and high temperature, and mediating air pollution was greater than high temperature. Furthermore, industrial level, important diseases (HIV, CVD, CLDC, NNSN, and MT) morbidity, an aging population rate, air pollution, and high temperature were associated with an increase in suicide mortality, whereas green coverage and income level were associated with a reduction in suicide rates. The findings demonstrate that appropriate urban policy and urban planning may lower suicide mortality, be useful strategies for suicide prevention, and be a foundation for building a healthy city. Moreover, this study provides clarity on the complex relationship of suicide and the urban environment while identifying crucial factors.

Analysis of the expression and prognostic value of MT1-MMP, ß1-integrin and YAP1 in glioma.

Increased expression of membrane type 1-matrix metalloproteinase (MT1-MMP/MMP14) is associated with the development of many cancers. MT1-MMP may promote the entry of yes-associated protein1 (YAP1) into the nucleus by regulating the regulation of ß1-integrin. The purpose of this study was to investigate the effects of MT1-MMP, ß1-integrin and YAP1 on the prognosis of gliomas. The expression of proteins was detected by bioinformatics and immunohistochemistry. The relationship between three proteins and clinicopathological parameters was analyzed by the χ 2 test. Survival analysis was used to investigate the effects of three proteins on prognosis. The results showed that high expressions of MT1-MMP, ß1-integrin and YAP1 were found in glioblastoma (GBM) compared with lower-grade glioma (LGG). There was a significantly positive correlation between MT1-MMP and ß1-integrin (r = 0.387), MT1-MMP and YAP1 (r = 0.443), ß1-integrin and YAP1 (r = 0.348). Survival analysis showed that patients with overexpression of MT1-MMP, ß1-integrin and YAP1 had a worse prognosis. YAP1 expression was the independent prognostic factor for progression-free survival (PFS). There was a statistical correlation between the expression of MT1-MMP and YAP1 and isocitrate dehydrogenase 1 (IDHl) mutation. Thus, this study suggested that MT1-MMP, ß1-integrin and YAP1, as tumor suppressors, are expected to be promising prognostic biomarkers and therapeutic targets for glioma patients.

Crucial factors of the built environment for mitigating carbon emissions.

Global warming and environmental changes are becoming increasingly threatened by carbon emissions, especially in urban areas. Low-carbon cities have the co-benefits of mediating environmental threats and lowering carbon emissions. However, the direct and indirect pathways and effects between the built environment and carbon emissions remain unclear, limiting low-carbon city development. Therefore, this study used partial least squares (PLS) modeling and urban-scale data from nineteen counties in Taiwan to identify the crucial effects and pathways affecting carbon emissions. The model considered the impacts of the characteristics of urban form (i.e., density, land mix, city size, urban sprawl, and jobs-housing balance), urban function (i.e., industrial and commercial levels), urban transportation, and urban greening on carbon emissions. The results reveal that minimizing city size, urban sprawl, industrial level, and transportation status, and maximizing density, land mix, commercial levels, and urban green coverage could reduce carbon emissions. This is the first study to apply PLS modeling to identify variable pathways and evaluate both direct and indirect effects of built environment characteristics on carbon emissions. Findings demonstrated that appropriate urban policies and planning, such as compact cities, green cities, or transit-oriented development, might lower carbon emissions and thus further serve as useful strategies for building low-carbon cities.

A Novel Classification Model for Lower-Grade Glioma Patients Based on Pyroptosis-Related Genes.

Recent studies demonstrated that pyroptosis plays a crucial role in shaping the tumor-immune microenvironment. However, the influence of pyroptosis on lower-grade glioma regarding immunotherapy and targeted therapy is still unknown. This study analyzed the variations of 33 pyroptosis-related genes in lower-grade glioma and normal tissues. Our study found considerable genetic and expression alterations in heterogeneity among lower-grade gliomas and normal brain tissues. There are two pyroptosis phenotypes in lower-grade glioma, and they exhibited differences in cell infiltration characteristics and clinical characters. Then, a PyroScore model using the lasso-cox method was constructed to measure the level of pyroptosis in each patient. PyroScore can refine the lower-grade glioma patients with a stratified prognosis and a distinct tumor immune microenvironment. Pyscore may also be an effective factor in predicting potential therapeutic benefits. In silico analysis showed that patients with a lower PyroScore are expected to be more sensitive to targeted therapy and immunotherapy. These findings may enhance our understanding of pyroptosis in lower-grade glioma and might help optimize risk stratification for the survival and personalized management of lower-grade glioma patients.

Separation and purification of highly infectious enterovirus A71 particles using a strong anion-exchange column.

Virions produced from cell culture is the primary source for production of formalin-inactivated whole virus vaccines for enteroviruses. EV-A71 particles produced from culture system comprise two major types, the immature/empty (E)-particle and the mature/full (F)-particle, which both exhibit low isoelectric point (pI) values but have distinct differences in infectivity and immunogenicity. Although EV-A71 particles can conventionally be separated into E-particle and F-particle using sucrose gradient ultracentrifugation, this procedure is cumbersome and difficult to put into practice for vaccine production. Methods based on ion-exchange chromatography have been exploited to improve the purification efficacy; however, none of them are capable of separating the E- and F-particles efficiently. In this study, we aimed to develop an approach to isolate and purify the highly immunogenic mature EV-A71 particles. By applying a step gradient elution procedure, we successfully isolated the viral structure protein VP0-cleaved particles of EV-A71 from a mixture of cultured viral solution using the Q-membrane anion-exchange chromatography. The elution started with 0.1x phosphate buffered saline (PBS) solution while increasing the percentage of 1x PBS containing 1M NaCl in sequential steps. By this procedure, the VP0-cleaved mature particles and VP0-uncleaved immature particles of EV-A71 could be separated into different fractions in Q-membrane with gradually increased NaCl concentration in elution buffer. The purified VP0-cleaved particles were shown to have characteristics equivalent to those of the highly infectious F-particles of EV-A71. The overall recovery rate for the mature EV-A71 particles by Q-membrane is 56% and its purity was shown to be equivalent to those isolated by the sucrose gradient ultracentrifugation. Our approach provides a simple and efficient purification method for recovering mature, highly infectious virus particles from the EV-A71 culture bulk.

A community-based study on associations between PM2.5 and PM1 exposure and heart rate variability using wearable low-cost sensing devices.

Few studies have investigated the effect of personal PM2.5 and PM1 exposures on heart rate variability (HRV) for a community-based population, especially in Asia. This study evaluates the effects of personal PM2.5 and PM1 exposure on HRV during two seasons for 35 healthy adults living in an urban community in Taiwan. The low-cost sensing (LCS) devices were used to monitor the PM levels and HRV, respectively, for two consecutive days. The mean PM2.5 and PM1 concentrations were 13.7 ± 11.4 and 12.7 ± 10.5 µg/m3 (mean ± standard deviation), respectively. Incense burning was the source that contributed most to the PM2.5 and PM1 concentrations, around 9.2 µg/m3, while environmental tobacco smoke exposure had the greatest impacts on HRV indices, being associated with the highest decrease of 20.2% for high-frequency power (HF). The results indicate that an increase in PM2.5 concentrations of one interquartile range (8.7 µg/m3) was associated with a change of -1.92% in HF and 1.60% in ratio of LF to HF power (LF/HF). Impacts on HRV for PM1 were similar to those for PM2.5. An increase in PM1 concentrations of one interquartile range (8.7 µg/m3) was associated with a change of -0.645% in SDNN, -1.82% in HF and 1.54% in LF/HF. Stronger immediate and lag effects of PM2.5 exposure on HRV were observed in overweight/obese subjects (body mass index (BMI) ≥24 kg/m2) compared to the normal-weight group (BMI <24 kg/m2). These results indicate that even low-level PM concentrations can still cause changes in HRV, especially for the overweight/obese population.

Analyzing protein-protein interactions in rare cells using microbead-based single-molecule pulldown assay.

For studying protein-protein interactions (PPIs) in general, a powerful and commonly used technique is conventional coimmunoprecipitation (co-IP/pulldown) followed by western blotting. However, the technique does not provide precise information regarding the kinetics and stoichiometry of PPIs. Another drawback is that the sensitivity of conventional co-IP is not suitable for examining PPIs in rare cells such as sensory hair cells, circulating tumor cells, embryonic stem cells, and subsets of immune cells. The current single-molecule pulldown (SiMPull) assay can potentially be used for studying PPIs in rare cells but its wide application is hindered by the high technical barrier and time consumption. We report an innovative, agarose microbead-based approach for SiMPull. We used commercially available, pre-surface-functionalized agarose microbeads to capture the protein of interest together with its binding partners specifically from cell extracts and observed these interactions under a microscope at the single-molecule level. Relative to the original method, microbead-based SiMPull is considerably faster, easier to use, and more reproducible and yet provides similar sensitivity and signal-to-background ratio; specifically, with the new method, sample-preparation time is substantially decreased (from ∼10 to ∼3 h). These crucial features should facilitate wide application of the powerful and versatile SiMPull method in common biological and clinical laboratories. Notably, by exploiting the simplicity and ultrahigh sensitivity of microbead-based SiMPull, we used the method in the study of rare auditory hair cells and γδ T cells for the first time.

Multiple impacts and pathways of urban form and environmental factors on cardiovascular mortality.

Air pollution and heat are significant threats to public health, especially in urban areas with intensive human activities under the trend of climate change. However, the mediation effects of urban form on health via air pollution and heat have been overlooked in previous investigations. This study explored the potential impacts and pathways of urban form on cardiovascular mortality through air pollutants and heat by using partial least squares model with data from Taiwan. The measurable characteristics of urban form include city size, urban sprawl, and mixed land use. Other factors that influence cardiovascular mortality, such as urban industrial level, economic status, aging population, and medical resource, were also considered in the model. Results revealed that maximizing mixed land use and minimizing city size and urban sprawl can help reduce cardiovascular mortality, and the minimizing city size was the most important one. Urban industrial level, economic status, aging population, and medical resource were also influential factors. This is the first study to consider the pathways and impacts of urban form on cardiovascular mortality, and our results indicate that proper urban planning and policy could reduce cardiovascular mortality.

Elevated methylation of cyclin dependent kinase inhibitor 2B contributes to the risk of coronary heart disease in women.

Cyclin dependent kinase inhibitor 2B (CDKN2B) encodes a cyclin-dependent kinase inhibitor that may enhance the formation of atherosclerotic plaques. The aim of the present study was to investigate the contribution of CDKN2B promoter methylation on the risk of coronary heart disease (CHD). The present results indicated a significant association between increased CDKN2B methylation and the risk of CHD (adjusted P=0.043). A breakdown analysis according to sex demonstrated that CDKN2B methylation was significantly associated with the risk of CHD in women (adjusted P=0.010), but not in men. A further breakdown analysis by age indicated a significant association of CHD in the women >60 years (P=0.024). Luciferase reporter gene assay results indicated that the CDKN2B promoter fragment significantly enhanced luciferase activity (P<0.001). In addition, CDKN2B transcription was significantly enhanced following treatment with 5-aza-2'-deoxycytidine methylation inhibitor in human aortic endothelial cells (HAEC) and human primary coronary artery smooth muscle cells (HPCASMC; P<0.05 and P<0.01), but not in 293 cells. Notably, estrogen treatment reduced CDKN2B methylation of several CpGs and significantly increased CDKN2B gene expression levels in HAEC, HPCASMC and 293 cells (P<0.05 and P<0.01). Additionally, treatment of HAEC and HPCASMC with simvastatin and γ-carboxy-L-glutamic acid reduced CDKN2B promoter methylation and increased CDKN2B transcription concomitantly. The present study suggests that CDKN2B promoter methylation may be associated with sex dimorphism in the pathogenesis of CHD.

Mediation pathways and effects of green structures on respiratory mortality via reducing air pollution.

Previous studies have shown both health and environmental benefits of green spaces, especially in moderating temperature and reducing air pollution. However, the characteristics of green structures have been overlooked in previous investigations. In addition, the mediation effects of green structures on respiratory mortality have not been assessed. This study explores the potential mediation pathways and effects of green structure characteristics on respiratory mortality through temperature, primary and secondary air pollutants separately using partial least squares model with data from Taiwan. The measurable characteristics of green structure include the largest patch percentage, landscape proportion, aggregation, patch distance, and fragmentation. The results showed that mortality of pneumonia and chronic lower respiratory diseases could be reduced by minimizing fragmentation and increasing the largest patch percentage of green structure, and the mediation effects are mostly through reducing air pollutants rather than temperature. Moreover, a high proportion of but fragmented green spaces would increase secondary air pollutants and enhance health risks; demonstrating the deficiency of traditional greening policy with primary focus on coverage ratio. This is the first research focusing on mediation effects of green structure characteristics on respiratory mortality, revealing that appropriate green structure planning can be a useful complementary strategy in environmental health management.