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Urban floods induced by the compound effects of climate change and rapid urbanisation are one of the key challenges facing many Chinese cities. In 2013, China initiated the Sponge City Program (SCP) to address this issue via constructing/restoring green-blue infrastructure as Nature-Based Solutions (NBS). Various green and blue spaces have been integrated into traditional grey facilities to mitigate urban floods, provide recreational venues, and enhance the natural aesthetics of urban landscapes. While housing development has been actively included in NBS provision, it remains unclear whether and how homebuying decisions might be influenced by NBS implementation under the framework of the Sponge City Program. Our study uses Guiyang, one of the pilots selected by the central government for the construction of “Sponge City” via implementing NBS. In-depth semi-structured interviews (n = 25) were used to examine whether the multiple benefits generated by newly established green-blue (sponge) facilities would affect residents’ home-purchasing preferences/decisions. We found that the provision of a green and blue facility, and whether it is part of the SCP or not, significantly influences a homebuyers’ choice. This study sheds additional light on the nexus of end users (homebuyers) and NBS provision (developers) under the framework of the Sponge City Program, generating practical implications for stakeholder engagement for NBS implementation to achieve sustainable urbanization.

Abstract Background Neuroblastoma, one of the most prevalent childhood cancers, is often treated with surgery, radiation, and chemotherapy. However, prognosis and survival are still dismal for children with neuroblastoma at high risk. Consequently, it is vital to identify new and effective treatment targets. As a component of the meiotic cohesion complex, REC8 is involved in a wide range of malignancies. The current work assessed the impact of REC8 knockdown on SH-SY5Y and SK-N-AS neuroblastoma cells and delved into the molecular mechanism behind this effect. Methods Knockdown of REC8 using the small interfering (si) RNA technology, and the results were verified by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and western blot. The Cell Counting Kit-8 (CCK-8) was used to examine cell proliferation, while flow cytometry was used to examine cell cycle progression and apoptosis. Analyses of angiogenesis included tube formation experiments. Transwell tests were used to examine cell migration and invasion. Results The data showed that downregulation of the REC8 led to a substantial decrease in cell proliferation by stopping the cell cycle in the G1 phase. REC8 knockdown significantly reduced neuroblastoma cell proliferation, migration, invasion, angiogenesis, induced cell cycle arrest, and enhanced apoptosis. We also discovered that repressing REC8 expression in neuroblastoma cell lines SH-SY5Y and SK-N-AS reduced their ability to activate the STAT3/VEGF signaling pathway. Conclusions Neuroblastoma therapy may benefit from targeting REC8 and its downstream targets.

Urban agriculture, the cultivation, processing and distribution of plants and animals within urban or suburban boundaries, is becoming an increasingly relevant strategy to enhance food safety for urban residents. With the rapid urbanization in the past decades, three forms of urban agriculture have emerged in China: small-scale, capital-intensive, and tourism-type. Each form presents unique benefits and challenges, and their development has been largely shaped by the local socio-economic context and government policies. The benefits of urban agriculture are numerous including the improvement of community cohesion, promotion of physical health, enhancement of environmental sustainability, and creation of new economic opportunities. However, it also faces many challenges such as land availability, capital shortage, market access, and governmental support. This review summarized the benefits of urban agriculture and its current limitations and challenges, and proposed strategies to overcome these hurdles in China based on successful examples in the Global North. Four specific recommendations were provided to promote urban agriculture in China: integration of urban agriculture into cities, solutions for land and resource use, government support and actions, as well as planning and regulations. Adoption of these recommendations is expected to result in healthier cities and a more robust agricultural system in China.

Abstract Background Grain commodities are important to people's daily lives and their fluctuations can cause instability for households. Accurate prediction of grain prices can improve food and social security. Methods & Materials This study proposes a hybrid Long Short‐Term Memory (LSTM)‐Convolutional Neural Network (CNN) model to forecast weekly oat, corn, soybean and wheat prices in the United States market. The LSTM‐CNN is a multivariate model that uses weather data, macroeconomic data, commodities grain prices and snow factors, including Snow Water Equivalent (SWE), snowfall and snow depth, to make multistep ahead forecasts. Results Of all the features, the snow factor is used for the first time for commodity price forecasting. We used the LSTM‐CNN model to evaluate the 5, 10, 15 and 20 weeks ahead forecasting and this hybrid model had the lowest Mean Squared Error (MSE) at 5, 10 and 15 weeks ahead of prediction. In addition, Shapley values were calculated to analyse the feature contribution of the LSTM‐CNN model when forecasting the testing set. Based on the feature contribution, SWE ranked third, fifth and seventh in feature importance in the 5‐week ahead forecast for corn, oats and wheat, respectively, and 7–8 places higher than total precipitation, indicating the potential use of SWE in grain price forecasting. Conclusion The hybrid multivariate LSTM‐CNN model outperformed other models and the newly involved climate data, SWE, showed the research potential of using snow as an input variable to predict grain prices over a multistep ahead time horizon.

ABSTRACTGlaciers in the Tibetan Plateau are melting at an unprecedented recently rate in the context of global warming. Time-sequenced landform mapping for the Hailuogou Glacier, a partly debris-covered glacier in the southeastern Tibetan Plateau, shows the detailed evolution of glacier changes as the ice recedes. This study presents four maps of the Hailuogou Glacier tongue, a partly debris-covered glacier in the southeastern Tibetan Plateau, documenting the spatial evolution of glaciological, hydrological, and geomorphological features from 2018 to 2021. Structure from Motion with Multiview Stereo software was applied to images captured by from uncrewed aerial vehicles were used to produce digital surface models and orthophoto mosaics. These datasets were used, and then to identify and map the features based on pre-defined mapping criteria. From 2018 to 2021, the glacier underwent continuous recession, with the terminus retreating, intense crevassing in the lower part of the ablation zone, and continuous expansion of the terminal disintegration area. The recent evolution of the glacier implies that the gradual disintegration of the lower glacier tongue is likely to be exacerbated over the next decades by the continuous climate warming.

Abstract Catchment floods are more challenging due to intensive urbanization and climate change. Enlightened by the Low Impact Development (LID), China initiated the Sponge City Program (SCP) to transform Urban Flood Management (UFM) to be more environmentally friendly in 2013. The China National Government (CNG) has subsidized municipal SCP facilities to enhance urban flood resilience while delivering multiple co‐benefits for urban ecosystems and social well‐being. Recent floods at Schleiden (Germany), Arizona State (USA), and Zhengzhou (China) in 2021 reflected the necessity of Catchment Flood Management (CFM) to cover the whole catchment scale. The SCP, designed to handle small‐scale urban pluvial floods, has brought concerns when facing larger‐scale fluvial floods after the Zhengzhou 2021 flood. Indeed, catchment‐scale Natural Flood Management (NFM) can manage fluvial floods while improving flood adaptations sustainably from upstream to downstream reaches. This research develops a new framework named the Sponge Catchment Management Plan (SCMP), including structural and Nonstructural elements. On the structural side, the SCMP framework integrates NFM with the SCP and Grey Engineering (GE) for reducing the fluvial flood discharge peaks in the whole‐catchment scale. On the nonstructural side, the SCMP encourages collaborative governance, revising technical standards, and improving “bottom‐up” participation. This research used Semi‐Structured Interviews (SSIs) and a Focus Group Approach (FGA) to explore 62 professional and Nonprofessional stakeholders’ perspectives on the SCMP framework. Some professional respondents did not know much about the NFM and were worried about the effectiveness of this practice. But most interviewees supported the SCMP pilot work and shared the co‐benefits from the NFM. This case study at the SW China, Guiyang, could be a lesson to encourage other Chinese cities further implement SCP to improve catchment‐scale flood resilience.

Hydropower reservoirs, as vital inland waters bodies of anthropogenic origin, exhibit distinct characteristics from natural waters, thereby garnering research interest in the quantification and mapping of greenhouse gas (GHG) emissions. In this review, we systematically examine studies focusing on GHG emissions from hydropower reservoirs. We identify two key primary physical mechanisms resulting from river damming, namely water impoundment and water regulation, which can significantly influence GHG emissions in hydropower reservoirs. Reservoirs vary in size, with smaller reservoirs exhibiting higher CH _4 emissions per unit area. For instance, small reservoirs have an average flux rate of 327.54 mg C–CH _4 /m ^2 /day, while medium-sized reservoirs emit 267.12 mg C–CH _4 /m ^2 /day, and large ones emit 37.34 mg C–CH _4 /m ^2 /day. This difference is potentially attributable to shorter water residence times in small reservoirs and increased susceptibility to littoral disturbance. In addition to reservoir scale, variations in GHG emissions between reservoirs are also influenced by the type of hydropower. Run-of-river and closed-loop pumped storage hydropower (PSH) systems are anticipated to exhibit lower GHG emissions (PSH: 4.2–46.5 mg C–CH _4 /m ^2 /day) in comparison to conventional impoundment hydropower, owing to their operational characteristics, facilitating mixing and oxygenation within the reservoir water column and reducing sedimentation. Nonetheless, further field measurements are warranted. Through the integration of literature insights, we propose solutions aimed at managing emissions, considering both physical mechanisms and hydropower planning. Ultimately, these findings will advance our understanding of GHG emissions from hydropower reservoirs and facilitate sustainable carbon reduction management practices.

IntroductionOil palm is the world's highest yielding oil crop and its palm oil has high nutritional value, making it an oilseed plant with important economic value and application prospects. After picking, oil palm fruits exposed to air will gradually become soft and accelerate the process of fatty acid rancidity, which will not only affect their flavor and nutritional value, but also produce substances harmful to the human body. As a result, studying the dynamic change pattern of free fatty acids and important fatty acid metabolism-related regulatory genes during oil palm fatty acid rancidity can provide a theoretical basis for improving palm oil quality and extending its shelf life.MethodsThe fruit of two shell types of oil palm, Pisifera (MP) and Tenera (MT), were used to study the changes of fruit souring at different times points of postharvesting, combined with LC-MS/MS metabolomics and RNA-seq transcriptomics techniques to analyze the dynamic changes of free fatty acids during fruit rancidity, and to find out the key enzyme genes and proteins in the process of free fatty acid synthesis and degradation according to metabolic pathways.Results and discussionMetabolomic study revealed that there were 9 different types of free fatty acids at 0 hours of postharvest, 12 different types of free fatty acids at 24 hours of postharvest, and 8 different types of free fatty acids at 36 hours of postharvest. Transcriptomic research revealed substantial changes in gene expression between the three harvest phases of MT and MP. Combined metabolomics and transcriptomics analysis results show that the expression of SDR, FATA, FATB and MFP four key enzyme genes and enzyme proteins in the rancidity of free fatty acids are significantly correlated with Palmitic acid, Stearic acid, Myristic acid and Palmitoleic acid in oil palm fruit. In terms of binding gene expression, the expression of FATA gene and MFP protein in MT and MP was consistent, and both were expressed higher in MP. FATB fluctuates unevenly in MT and MP, with the level of expression growing steadily in MT and decreasing in MP before increasing. The amount of SDR gene expression varies in opposite directions in both shell types. The above findings suggest that these four enzyme genes and enzyme proteins may play an important role in regulating fatty acid rancidity and are the key enzyme genes and enzyme proteins that cause differences in fatty acid rancidity between MT and MP and other fruit shell types. Additionally, differential metabolite and differentially expressed genes were present in the three postharvest times of MT and MP fruits, with the difference occurring 24 hours postharvest being the most notable. As a result, 24 hours postharvest revealed the most obvious difference in fatty acid tranquility between MT and MP shell types of oil palm. The results from this study offer a theoretical underpinning for the gene mining of fatty acid rancidity of various oil palm fruit shell types and the enhancement of oilseed palm acid-resistant germplasm cultivation using molecular biology methods.

Recent evidence suggests that lung microbiota can be recognized as one of the ecological determinants of various respiratory diseases. However, alterations in the lung microbiota and associated lung immunity in these respiratory diseases remain unclear. To compare the lung microbiota and lung immune profiles in common respiratory diseases, a total of 78 patients were enrolled in the present study, including 21 patients with primary pulmonary tuberculosis (PTB), eight patients with newly diagnosed lung cancer (LC), and 49 patients with community-acquired pneumonia (CAP). Bronchoalveolar lavage fluid (BALF) was collected for microbiota and cytokine analyses. With MiSeq sequencing system, increased bacterial alpha-diversity and richness were observed in patients with LC than in those with PTB and CAP. Linear discriminant analysis effect size revealed that CAP-associated pulmonary microbiota were significantly different between the PTB and LC groups. More key functionally different genera were found in the PTB and LC groups than in the CAP group. The interaction network revealed stronger positive and negative correlations among these genera in the LC group than in the other two groups. However, increased BALF cytokine profiles were observed in the PTB group than in the other two groups, while BALF cytokines were correlated with key functional bacteria. This comparative study provides evidence for the associations among altered lung microbiota, BALF inflammation, and different respiratory disorders, which provides insight into the possible roles and mechanisms of pulmonary microbiota in the progression of respiratory disorders.

The fruit of the oil palm (Elaeis guineensis Jacq.) has fleshy mesocarpic tissue rich in lipids. This edible vegetable oil is economically and nutritionally significant across the world. The core concepts of oil biosynthesis in oil palms remain to be researched as the knowledge of oil biosynthesis in plants improves. In this study, we utilized a metabolite approach and mass spectral analysis to characterize metabolite changes and identify the sequences of protein accumulation during the physiological processes that regulate oil synthesis during oil palm fruit ripening. Here, we performed a comprehensive lipidomic data analysis in order to understand the role of lipid metabolism in oil biosynthesis mechanisms. The experimental materials were collected from the mesocarp of oil palm (Tenera) at 95 days (early accumulation of fatty acid, first stage), 125 days (rapid growth of fatty acid accumulation, second stage), and 185 days (stable period of fatty acid accumulation, third stage) after pollination. To gain a clear understanding of the lipid changes that occurred during the growth of the oil palm, the metabolome data were found using principal component analysis (PCA). Furthermore, the accumulations of diacylglycerols, ceramides, phosphatidylethanolamine, and phosphatidic acid varied between the developmental stages. Differentially expressed lipids were successfully identified and functionally classified using KEGG analysis. Proteins related to the metabolic pathway, glycerolipid metabolism, and glycerphospholipid metabolism were the most significantly changed proteins during fruit development. In this study, LC-MS analysis and evaluation of the lipid profile in different stages of oil palm were performed to gain insight into the regulatory mechanisms that enhance fruit quality and govern differences in lipid composition and biosynthesis.

Rapid urbanization has sharply increased the pressure of urban water issues (e.g., urban flooding and water pollution) in the Chinese megacities during last three decades. Sustainable urban water management approaches, such as Nature Based Solutions (NBS) and Low Impact Developments (LIDs), have successfully delivered long-term benefits to cities in Europe and North America. Similarly, the Chinese Sponge City Program (SCP) initiated in 2013 and experimented in 30 pilot Chinese cities. This paper reviewed the first stage of the SCP from 2015 to 2020 by using observation, Semi-Structured Interview (SSI) and Focus Group Approach (FGA) in context of Gui'an New Distinct, SW China to investigate the SCP progress and issues: (1). whether it is effectively solving urban water issues; (2). if there are any major challenges and barriers in the SCP practice. The findings of this case study provide potential solutions for improving the SCP and sustainable urban water management and possibly transform to other Sponge Cities in China.

Objective: The coexistence of maternal malignancy and pregnancy has received increasing attention in Noninvasive prenatal testing (NIPT) studies. Malignancy in pregnant women potentially affects the copy number variation (CNV) profile in NIPT results. Only one case of hematologic cancer has been reported in a Hong-Kong pregnant women, and solid tumors have never been reported in pregnant Chinese women. Case report: The patients with dysgerminoma and cervical cancer showed aberrant chromosomal aneuploidies in NIPT and concordant patterns of genome disruption in tumor tissues. The genomic aberrations in the gastric cancer patient had similar copy number variation pattern of gastric cancer. Conclusion: The findings in this study and the literature review further validate the effect of maternal malignancy on the copy number variation profile in NIPT data and strengthen the possibility of detecting malignant tumors with NIPT in the future. Keywords: Noninvasive prenatal testing (NIPT), Copy number variation (CNV) profile, Massively parallel sequencing, Maternal malignancy, Multiple aneuploidies

Abstract Background The green peach aphid (GPA), Myzus persicae (Sülzer), is a widespread phloem-feeding insect that significantly influences the yield and visual quality of peach [Prunus persica (L.) Batsch]. Single dominant gene (Rm3)-based resistance provides effective management of this invasive pest, although little is known about the molecular responses of plants to GPA feeding. Results To illustrate the molecular mechanisms of monogenic resistance in peach to young tissue-infecting GPAs, aphid-resistant/aphid-susceptible peach lines from a segregating population with Rm3/rm3 and rm3/rm3 genotypes were infested with GPAs for 3 to 72 h. Transcriptome analysis of the infested tissues identified 3854 differentially expressed genes (DEGs). Although the majority of the DEGs in the resistant line also responded to aphid attack in the susceptible line, the overall magnitude of change was greater in the resistant line than in the susceptible line. The enriched gene ontology of the 3854 DEGs involved in plant defence responses included redox situation, calcium-mediated signalling, transcription factor (e.g., WRKY, MYB, and ERF), MAPK signalling cascade, phytohormone signalling, pathogenesis-related protein, and secondary metabolite terms. Of the 53 genes annotated in a 460 kb interval of the rm3 locus, seven genes were differentially expressed between the aphid-resistant and aphid-susceptible peach lines following aphid infestation. Conclusions Together, these results suggest that the Rm3-dependent resistance relies mainly on the inducible expression of defence-related pathways and signalling elements within hours after the initiation of aphid feeding and that the production of specific secondary metabolites from phenylpropanoid/flavonoid pathways can have major effects on peach-aphid interactions.

TIM-1 is a critical gene that regulates T-helper cell development. However, little research has revealed the distribution, prognosis, and immune infiltration of TIM-1 in cancers. TCGA, GEO, Oncomine, TIMER, Kaplan-Meier, PrognoScan, GEPIA, TISIDB, and HPA databases were used to analyze TIM-1 in cancers. High TIM-1 expression was observed in bladder, cholangio, head and neck, colorectal, gastric, kidney, liver, lung adenocarcinoma, skin, uterine corpus endometrial, and pancreatic cancers compared to the normal tissues, and immunofluorescence shows that TIM-1 is mainly localized in vesicles. Simultaneously, high TIM-1 expression was closely related with poorer overall survival in gastric, lung adenocarcinoma, and poorer disease-specific survival in gastric cancer in the TCGA cohort, and was validated in the GEO cohort. Moreover, high expression of TIM-1, correlated with clinical relevance of gastric cancer and lung adenocarcinoma, was associated with tumor-infiltrating lymphocytes in lung adenocarcinoma and gastric cancer. Finally, immunohistochemistry showed TIM-1 expression was higher in lung adenocarcinoma and gastric cancer compared to the normal tissues. In summary, we applied integrated bioinformatics approaches to suggest that TIM-1 can be used as a prognostic biomarker in gastric and lung adenocarcinoma, which might provide a novel direction to explore the pathogenesis of gastric and lung adenocarcinoma.

Flood management is a complex issue in Chinese cities that exhibit high populations and have undergone rapid urbanization. Urban flood management (UFM) approaches can be used to mitigate urban flood risk. To address urban issues of poor water quality and urban surface flooding, the Sponge City Program (SCP) was initiated in 2013 in China. The SCP aims to provide an opportunity for Chinese cities to improve their current UFM practices. This study looks at Guiyang (a pilot sponge city located in SW China) as a case study to identify the challenges and opportunities of UFM in China. Guiyang is a valley city surrounded by a hilly landscape. Using interview records and flood data, we illustrate that the primary type of flood in Guiyang is fluvial rather than surface water flooding. In Guiyang, the current function and targets of the SCP have yet to engage with the catchment level flood management, instead mainly focusing on the site-specific context (i.e., community level). Catchment flood management planning (CFMP) and natural flood management (NFM) both address this problem and may be a more suitable approach to manage flood discharge from the upper and middle catchments in Guiyang. In addition, it is suggested that a mixed option combining “hard” infrastructure (e.g., reservoirs and floodwalls) with “soft” flood management measures (e.g., improving people awareness and participation) may improve urban flood resilience in Chinese cities.

Fine particulate matter in the lower atmosphere (PM2.5) continues to be a major public health problem globally. Identifying the key contributors to PM2.5 pollution is important in monitoring and managing atmospheric quality, for example, in controlling haze. Previous research has been aimed at quantifying the relationship between PM2.5 values and their underlying factors, but the spatial and temporal dynamics of these factors are not well understood. Based on random forest and Shapley additive explanation (SHAP) algorithms, this study analyses the spatiotemporal variations in selected key factors influencing PM2.5 in Zhejiang Province, China, for the period 2000–2019. The results indicate that, while factors influencing PM2.5 varied significantly during the period studied, SHAP values suggest that there is consistency in their relative importance as follows: meteorological factors (e.g., atmospheric pressure) > socioeconomic factors (e.g., gross domestic product, GDP) > topography and land cover factors (e.g., elevation). The contribution of GDP and transportation factors initially increased but has declined in the recent past, indicating that economic and infrastructural development does not necessarily result in increased PM2.5 concentrations. Vegetation productivity, as indicated by changes in NDVI, is demonstrated to have become more important in improving air quality, and the area of the province over which it constrains PM2.5 concentrations has increased between 2000 and 2019. Mapping of SHAP values suggests that, although the relative importance of industrial emissions has declined during the period studied, the actual area positively impacted by such emissions has actually increased. Despite developments in government policy, greater efforts to conserve energy and reduce emissions are still needed. The study further demonstrates that the combination of random forest and SHAP methods provides a valuable means to identify regional differences in key factors affecting atmospheric PM2.5 values and offers a reliable reference for pollution control strategies.

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Urban flooding has become a serious issue in most Chinese cities due to rapid urbanization and extreme weather, as evidenced by severe events in Beijing (2012), Ningbo (2013), Guangzhou (2015), Wuhan (2016), Shenzhen (2019), and Chongqing (2020). The Chinese “Sponge City Program” (SCP), initiated in 2013 and adopted by 30 pilot cities, is developing solutions to manage urban flood risk, purify stormwater, and provide water storage opportunities for future usage. Emerging challenges to the continued implementation of Sponge Cities include (1) uncertainty regarding future hydrological conditions related to climate change projections, which complicates urban planning and designing infrastructure that will be fit for purpose over its intended operating life, and (2) the competing priorities of stakeholders and their reluctance to make trade-offs, which obstruct future investment in the SCP. Nature-Based Solutions (NBS) is an umbrella concept that emerged from Europe, which encourages the holistic idea of considering wider options that combine “Blue–Green” practices with traditional engineering to deliver “integrated systems of Blue–Green–Grey infrastructure”. NBS includes interventions making use of natural processes and ecosystem services for functional purposes, and this could help to improve current pilot SCP practices. This manuscript reviews the development of the SCP, focusing on its construction and design aspects, and discusses how approaches using NBS could be included in the SCP to tackle not only urban water challenges but also a wide range of social and environmental challenges, including human health, pollution (via nutrients, metals, sediments, plastics, etc.), flood risk, and biodiversity.

Targeted protein degradation (TPD) mediates protein level through small molecule induced redirection of E3 ligases to ubiquitinate neo-substrates and mark them for proteasomal degradation. TPD has recently emerged as a key modality in drug discovery. So far only a few ligases have been utilized for TPD. Interestingly, the workhorse ligase CRBN has been observed to be downregulated in settings of resistance to immunomodulatory inhibitory drugs (IMiDs). Here we show that the essential E3 ligase receptor DCAF1 can be harnessed for TPD utilizing a selective, non-covalent DCAF1 binder. We confirm that this binder can be functionalized into an efficient DCAF1-BRD9 PROTAC. Chemical and genetic rescue experiments validate specific degradation via the CRL4 DCAF1 E3 ligase. Additionally, a dasatinib-based DCAF1 PROTAC successfully degrades cytosolic and membrane-bound tyrosine kinases. A potent and selective DCAF1-BTK-PROTAC (DBt-10) degrades BTK in cells with acquired resistance to CRBN-BTK-PROTACs while the DCAF1-BRD9 PROTAC (DBr-1) provides an alternative strategy to tackle intrinsic resistance to VHL-degrader, highlighting DCAF1-PROTACS as a promising strategy to overcome ligase mediated resistance in clinical settings., (© 2024. The Author(s).)

RAS-MAPK signalling is fundamental for cell proliferation and is altered in most human cancers 1-3 . However, our mechanistic understanding of how RAS signals through RAF is still incomplete. Although studies revealed snapshots for autoinhibited and active RAF-MEK1-14-3-3 complexes 4 , the intermediate steps that lead to RAF activation remain unclear. The MRAS-SHOC2-PP1C holophosphatase dephosphorylates RAF at serine 259, resulting in the partial displacement of 14-3-3 and RAF-RAS association 3,5,6 . MRAS, SHOC2 and PP1C are mutated in rasopathies-developmental syndromes caused by aberrant MAPK pathway activation 6-14 -and SHOC2 itself has emerged as potential target in receptor tyrosine kinase (RTK)-RAS-driven tumours 15-18 . Despite its importance, structural understanding of the SHOC2 holophosphatase is lacking. Here we determine, using X-ray crystallography, the structure of the MRAS-SHOC2-PP1C complex. SHOC2 bridges PP1C and MRAS through its concave surface and enables reciprocal interactions between all three subunits. Biophysical characterization indicates a cooperative assembly driven by the MRAS GTP-bound active state, an observation that is extendible to other RAS isoforms. Our findings support the concept of a RAS-driven and multi-molecular model for RAF activation in which individual RAS-GTP molecules recruit RAF-14-3-3 and SHOC2-PP1C to produce downstream pathway activation. Importantly, we find that rasopathy and cancer mutations reside at protein-protein interfaces within the holophosphatase, resulting in enhanced affinities and function. Collectively, our findings shed light on a fundamental mechanism of RAS biology and on mechanisms of clinically observed enhanced RAS-MAPK signalling, therefore providing the structural basis for therapeutic interventions., (© 2022. The Author(s).)

SYNGAP1 is a neuronal Ras and Rap GTPase-activating protein with important roles in regulating excitatory synaptic plasticity. While many SYNGAP1 missense and nonsense mutations have been associated with intellectual disability, epilepsy, schizophrenia, and autism spectrum disorder (ASD), whether and how they contribute to individual disease phenotypes is often unknown. Here, we characterize 57 variants in seven assays that examine multiple aspects of SYNGAP1 function. Specifically, we used multiplex phospho-flow cytometry to measure variant impact on protein stability, pERK, pGSK3β, pp38, pCREB, and high-content imaging to examine subcellular localization. We find variants ranging from complete loss-of-function (LoF) to wild-type (WT)-like in their regulation of pERK and pGSK3β, while all variants retain at least partial ability to dephosphorylate pCREB. Interestingly, our assays reveal that a larger proportion of variants located within the disordered domain of unknown function (DUF) comprising the C-terminal half of SYNGAP1 exhibited higher LoF, compared to variants within the better studied catalytic domain. Moreover, we find protein instability to be a major contributor to dysfunction for only two missense variants, both located within the catalytic domain. Using high-content imaging, we find variants located within the C2 domain known to mediate membrane lipid interactions exhibit significantly larger cytoplasmic speckles than WT SYNGAP1. Moreover, this subcellular phenotype shows both correlation with altered catalytic activity and unique deviation from signaling assay results, highlighting multiple independent molecular mechanisms underlying variant dysfunction. Our multidimensional dataset allows clustering of variants based on functional phenotypes and provides high-confidence, multi-functional measures for making pathogenicity predictions., (Copyright © 2020 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Advances in sequencing technology have led to an explosion in the number of known genetic variants of human genes. A major challenge is to now determine which of these variants contribute to diseases as a result of their effect on gene function. Here, we describe a generic approach using the yeast Saccharomyces cerevisiae to quickly develop gene-specific in vivo assays that can be used to quantify the level of function of a genetic variant. Using synthetic dosage lethality screening, 'sentinel' yeast strains are identified that are sensitive to overexpression of a human disease gene. Variants of the gene can then be functionalized in a high-throughput fashion through simple growth assays using solid or liquid media. Sentinel interaction mapping (SIM) has the potential to create functional assays for the large majority of human disease genes that do not have a yeast orthologue. Using the tumour suppressor gene PTEN as an example, we show that SIM assays can provide a fast and economical means to screen a large number of genetic variants., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

As sequencing becomes more economical, we are identifying sequence variations in the population faster than ever. For disease-associated genes, it is imperative that we differentiate a sequence variant as either benign or pathogenic, such that the appropriate therapeutic interventions or surveillance can be implemented. PTEN is a frequently mutated tumor suppressor that has been linked to the PTEN hamartoma tumor syndrome. Although the domain structure of PTEN and the functional impact of a number of its most common tumor-linked mutations have been characterized, there is a lack of information about many recently identified clinical variants. To address this challenge, we developed a cell-based assay that utilizes a premalignant phenotype of normal mammary epithelial cells lacking PTEN. We measured the ability of PTEN variants to rescue the spheroid formation phenotype of PTEN -/- MCF10A cells maintained in suspension. As proof of concept, we functionalized 47 missense variants using this assay, only 19 of which have clear classifications in ClinVar. We utilized a machine learning model trained with annotated genotypic data to classify variants as benign or pathogenic based on our functional scores. Our model predicted with high accuracy that loss of PTEN function was indicative of pathogenicity. We also determined that the pathogenicity of certain variants may have arisen from reduced stability of the protein product. Overall, this assay outperformed computational predictions, was scalable, and had a short run time, serving as an ideal alternative for annotating the clinical significance of cancer-associated PTEN variants. SIGNIFICANCE: Combined three-dimensional tumor spheroid modeling and machine learning classifies PTEN missense variants, over 70% of which are currently listed as variants of uncertain significance. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/13/2775/F1.large.jpg., (©2020 American Association for Cancer Research.)

Functional variomics provides the foundation for personalized medicine by linking genetic variation to disease expression, outcome and treatment, yet its utility is dependent on appropriate assays to evaluate mutation impact on protein function. To fully assess the effects of 106 missense and nonsense variants of PTEN associated with autism spectrum disorder, somatic cancer and PTEN hamartoma syndrome (PHTS), we take a deep phenotypic profiling approach using 18 assays in 5 model systems spanning diverse cellular environments ranging from molecular function to neuronal morphogenesis and behavior. Variants inducing instability occur across the protein, resulting in partial-to-complete loss-of-function (LoF), which is well correlated across models. However, assays are selectively sensitive to variants located in substrate binding and catalytic domains, which exhibit complete LoF or dominant negativity independent of effects on stability. Our results indicate that full characterization of variant impact requires assays sensitive to instability and a range of protein functions.

A major challenge facing the genetics of autism spectrum disorders (ASDs) is the large and growing number of candidate risk genes and gene variants of unknown functional significance. Here, we used Caenorhabditis elegans to systematically functionally characterize ASD-associated genes in vivo. Using our custom machine vision system, we quantified 26 phenotypes spanning morphology, locomotion, tactile sensitivity, and habituation learning in 135 strains each carrying a mutation in an ortholog of an ASD-associated gene. We identified hundreds of genotype-phenotype relationships ranging from severe developmental delays and uncoordinated movement to subtle deficits in sensory and learning behaviors. We clustered genes by similarity in phenomic profiles and used epistasis analysis to discover parallel networks centered on CHD8•chd-7 and NLGN3•nlg-1 that underlie mechanosensory hyperresponsivity and impaired habituation learning. We then leveraged our data for in vivo functional assays to gauge missense variant effect. Expression of wild-type NLG-1 in nlg-1 mutant C. elegans rescued their sensory and learning impairments. Testing the rescuing ability of conserved ASD-associated neuroligin variants revealed varied partial loss of function despite proper subcellular localization. Finally, we used CRISPR-Cas9 auxin-inducible degradation to determine that phenotypic abnormalities caused by developmental loss of NLG-1 can be reversed by adult expression. This work charts the phenotypic landscape of ASD-associated genes, offers in vivo variant functional assays, and potential therapeutic targets for ASD., Competing Interests: The authors declare no competing interest.

With rare exception, ciliated cells entering mitosis lose their cilia, thereby freeing basal bodies to serve as centrosomes in the formation of high-fidelity mitotic spindles. Cilia can be lost by shedding or disassembly, but either way, it appears that the final release may be via a coordinated severing of the nine axonemal outer doublet microtubules linking the basal body to the ciliary transition zone. Little is known about the mechanism or regulation of this important process. The stress-induced deflagellation response of Chlamydomonas provides a basis to identifying key players in axonemal severing. In an earlier screen we uncovered multiple alleles for each of three deflagellation genes, ADF1, FA1, and FA2 Products of the two FA genes localize to the site of axonemal severing and encode a scaffolding protein and a member of the NIMA-related family of ciliary-cell cycle kinases. The identity of the ADF1 gene remained elusive. Here, we report a new screen using a mutagenesis that yields point mutations in Chlamydomonas, an enhanced screening methodology, and whole genome sequencing. We isolated numerous new alleles of the three known genes, and one or two alleles each of at least four new genes. We identify ADF1 as a TRP ion channel, which we suggest may reside at the flagellar transition zone., (Copyright © 2016 Hilton et al.)