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Exosomes, small membrane vesicles （30-100 nm） of endocytic origin secreted by most cell types, contain functional biomolecules, which can be horizontally transferred to recipient cells [1]. Exosomes bear a specific protein and lipid composition, and carry a select set of functional mRNAs and microRNAs [2]. Recently, our group has shown that c-Met shed in exosomes can promote a proangiogenic and prometastatic phenotype in bone marrow-derived progenitor cells during melanoma progression [3]. In previous research, retrotransposon RNA transcripts, single-stranded DNA （ssDNA）,

By virtue of their accumulated genetic alterations, tumor cells may acquire vulnerabilities that create opportunities for therapeutic intervention. We have devised a massively parallel strategy for screening short hairpin RNA (shRNA) collections for stable loss-of-function phenotypes. We assayed from 6000 to 20,000 shRNAs simultaneously to identify genes important for the proliferation and survival of five cell lines derived from human mammary tissue. Lethal shRNAs common to these cell lines targeted many known cell-cycle regulatory networks. Cell line-specific sensitivities to suppression of protein complexes and biological pathways also emerged, and these could be validated by RNA interference (RNAi) and pharmacologically. These studies establish a practical platform for genome-scale screening of complex phenotypes in mammalian cells and demonstrate that RNAi can be used to expose genotype-specific sensitivities.

While the Polycomb complex is known to regulate cell identity in ES cells, its role in controlling tissue‐specific stem cells is not well understood. Here we show that removal of Ezh1 and Ezh2, key Polycomb subunits, from mouse skin results in a marked change in fate determination in epidermal progenitor cells, leading to an increase in the number of lineage‐committed Merkel cells, a specialized subtype of skin cells involved in mechanotransduction. By dissecting the genetic mechanism, we showed that the Polycomb complex restricts differentiation of epidermal progenitor cells by repressing the transcription factor Sox2. Ablation of Sox2 results in a dramatic loss of Merkel cells, indicating that Sox2 is a critical regulator of Merkel cell specification. We show that Sox2 directly activates Atoh1 , the obligate regulator of Merkel cell differentiation. Concordantly, ablation of Sox2 attenuated the Ezh1/2 ‐null phenotype, confirming the importance of Polycomb‐mediated repression of Sox2 in maintaining the epidermal progenitor cell state. Together, these findings define a novel regulatory network by which the Polycomb complex maintains the progenitor cell state and governs differentiation in vivo . Ezh1/2‐mediated repression of the transcription factor Sox2 in epidermal progenitors extends the role of Polycomb proteins in controlling cellular identity beyond ES cells towards tissue‐specific stem cells.

Endometrial cancer is the most frequent type of cancer in the female reproductive tract. Loss-of-function alterations in PTEN, leading to enhanced PI3K/AKT activation, are among the most frequent molecular alterations in endometrial cancer. Increased PI3K/AKT signaling resulting from PTEN loss promotes cellular proliferation and confers resistance to TGFβ-mediated apoptosis, a key regulator of endometrial homeostasis. In this study, we have analyzed the role of miRNAs in driving these altered cellular responses. A comprehensive transcriptomic analysis of miRNA expression revealed the upregulation of several miRNAs caused by PTEN deficiency and/or TGFβ stimulation. The miR-424(322) ~ 503 cluster drew our attention due to its involvement in regulating apoptosis and proliferation. However, miR-424(322) ~ 503 cluster has a paradoxical role in cancer, exhibiting either oncogenic and tumor suppressive functions depending on cell type or context. To ascertain the function of miR-424(322) ~ 503 in endometrial carcinogenesis caused by PTEN deficiency, we generated a double Pten/miR-424(322) ~ 503 knock-out mice. We demonstrate that loss of miR-424(322) ~ 503 impairs proliferation of both wild type or Pten deficient endometrial organoids by interfering with growth factor and PI3K/AKT signaling. Furthermore, the absence of miR-424(322) ~ 503 restores TGFβ-induced apoptosis, which is otherwise compromised by PTEN deficiency. In vivo, Pten /miR-424(322) ~ 503 knock-out mice exhibit reduced endometrial cancer progression compared to Pten deficient mice through a cell-autonomous mechanism.

Despite the identification of MYCN amplification as an adverse prognostic marker in neuroblastoma, MYCN inhibitors have yet to be developed. Here, by integrating evidence from a whole-genome shRNA library screen and the computational inference of master regulator proteins, we identify transcription factor activating protein 4 (TFAP4) as a critical effector of MYCN amplification in neuroblastoma, providing a novel synthetic lethal target. We demonstrate that TFAP4 is a direct target of MYCN in neuroblastoma cells, and that its expression and activity strongly negatively correlate with neuroblastoma patient survival. Silencing TFAP4 selectively inhibits MYCN- amplified neuroblastoma cell growth both in vitro and in vivo, in xenograft mouse models. Mechanistically, silencing TFAP4 induces neuroblastoma differentiation, as evidenced by increased neurite outgrowth and upregulation of neuronal markers. Taken together, our results demonstrate that TFAP4 is a key regulator of MYCN -amplified neuroblastoma and may represent a valuable novel therapeutic target.

Lung cancer has been the most common cancer worldwide for several decades. The outcomes of patients with locally advanced lung cancer remain dismal, and only a minority of patients survive more than 5 years. However, tumor therapeutic resistance mechanisms are poorly studied. Identification of therapeutic resistance pathways in lung cancer in order to increase the sensitivity of lung tumor cells to therapeutic agents is a crucial but challenging need. To identify novel genes that modulate the response to platinum-based therapy, we performed a genome-wide high-throughput ribonucleic acid interference (RNAi) screen via transfection of human lung cancer (PC9) cells with a viral short hairpin RNA (shRNA) library. We further validated a potential target via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and clonogenic survival assays on PC9 and A549 lung tumor cells transfected with small interfering RNAs (siRNAs) to successfully downregulate protein expression and then treated with increasing doses of cisplatin or X-ray radiation. We determined protein expression by immunohistochemistry (IHC) after chemoradiotherapy and analyzed gene expression-based survival outcomes in two cohorts of human non-small-cell lung cancer (NSCLC) patients. The screen identified several targets involved in epithelial-to-mesenchymal transition (EMT), including Smurf1, Smurf2, YAP1, and CEBPZ, and glycolytic pathway proteins, including PFKFB3. Furthermore, we found that the small molecule proteasome inhibitor bortezomib significantly downregulated Smurf2 in lung cancer cells. The addition of bortezomib in combination with cisplatin and radiation therapy in PC9 and A549 cells led to an increase in deoxyribonucleic acid (DNA) double-strand breaks with increased numbers of γ-H2AX-positive cells and upregulation of apoptosis. Finally, we found that Smurf2 protein expression was upregulated in situ after treatment with cisplatin and radiation therapy in a relevant cohort of patients with stage III NSCLC. Additionally, Smurf2 gene expression was the strongest predictor of survival in patients with squamous NSCLC after chemotherapy or chemoradiotherapy. We successfully identified and validated Smurf2 as both a common modulator of resistance and an actionable target in lung cancer. These results suggest the urgent need to investigate clinical Smurf2 inhibition via bortezomib in combination with cisplatin and radiation for patients with locally advanced NSCLC.

The expansion of Urban Air Mobility (UAM) has led to diverse aircraft designs, with piloted systems expected to evolve into remotely piloted and automated operations. Future advancements in Intelligent Transportation Systems (ITSs) will further improve automation capabilities, promising significant benefits to the environment and overall efficiency of UAM aircraft. However, UAM aircraft face unique operational conditions that need to be accounted for when assessing safety risks, such as lower operating altitudes and hazards present in urban settings, thus leading to a potential increased risk of collisions with foreign objects, particularly birds and drones. This paper reviews historical safety data with an aim to better assess the potential risks of UAM aircraft. A survey was conducted to gather quantitative and qualitative insights from subject matter experts, reinforcing findings from existing studies. The results highlight the need for a comprehensive risk assessment framework to guide design improvements and regulatory strategies, ensuring safer UAM operations.

Loss-of-function phenotypes often hold the key to understanding the connections and biological functions of biochemical pathways. We and others previously constructed libraries of short hairpin RNAs that allow systematic analysis of RNA interference–induced phenotypes in mammalian cells. Here we report the construction and validation of second-generation short hairpin RNA expression libraries designed using an increased knowledge of RNA interference biochemistry. These constructs include silencing triggers designed to mimic a natural microRNA primary transcript, and each target sequence was selected on the basis of thermodynamic criteria for optimal small RNA performance. Biochemical and phenotypic assays indicate that the new libraries are substantially improved over first-generation reagents. We generated large-scale-arrayed, sequence-verified libraries comprising more than 140,000 second-generation short hairpin RNA expression plasmids, covering a substantial fraction of all predicted genes in the human and mouse genomes. These libraries are available to the scientific community.

Engine failure after take-off (or one engine being inoperative) is an exercise conducted as part of multi-engine flight training and on-going competency checking. To prepare pilots to manage a real in-flight emergency, this exercise has traditionally been conducted immediately after take-off. This has led to increased risks of fatal accidents due to the reduced height at which these exercises are typically conducted. Yet, there is variation in the heights stipulated in training procedures published by different stakeholders worldwide. Additionally, the conduct of the exercise has resulted in fatal accidents worldwide. This paper aims to review the previous literature on aviation training and aviation occurrence data to determine what empirical data exists to support the method of conducting simulated engine failures. Peer-reviewed academic publications on aviation training, aviation occurrence databases such as aviation investigation reports, and guidance materials published by aviation authorities on simulated training exercises will be included in this paper. It was found that the previous research on these exercises has focused on the transfer of motion cues or pilot responses to abnormal situations, but did not include specific data comparing pilot performance at different heights above ground level. A review of aviation occurrences found that actual engine failures occurred at higher heights that those used in simulated engine failures. A comparison of the guidance published by aviation authorities identified variations in the minimum altitude published and differing justifications for the minimum height chosen. Future research is needed to compare pilot performance during simulated engine failures to determine the ideal height to conduct the exercise to be representative of an actual engine failure while maintaining safety margins.

The Brazilian Amazon is the world's largest rainforest regions and plays a key role in biodiversity conservation as well as climate adaptation and mitigation. The government has created a network of protected areas (PAs) to ensure long-term conservation of the region. However, despite the importance of and positive advances in the establishment of PAs, natural resource depletion in the Brazilian Amazon is pervasive. We evaluated a total of 4,243 official law enforcement records generated between 2010 and 2015 to understand the geographical distribution of the illegal use of resources in federal PAs in the Brazilian Amazon. We classified illegal activities into ten categories and used generalized additive models (GAMs) to evaluate the relationship between illegal use of natural resources inside PAs with management type, age of PAs, population density, and accessibility. We found 27 types of illegal use of natural resources that were grouped into 10 categories of illegal activities. Most infractions were related to suppression and degradation of vegetation (37.40%), followed by illegal fishing (27.30%) and hunting activities (18.20%). The explanatory power of the GAMs was low for all categories of illegal activity, with a maximum explained variation of 41.2% for illegal activities as a whole, and a minimum of 14.6% for hunting activities. These findings demonstrate that even though PAs are fundamental for nature conservation in the Brazilian Amazon, the pressures and threats posed by human activities include a broad range of illegal uses of natural resources. Population density up to 50 km from a PA is a key variable, influencing illegal activities. These threats endanger long-term conservation and many efforts are still needed to maintain PAs that are large enough and sufficiently intact to maintain ecosystem functions and protect biodiversity.