An optimized protocol for the generation and monitoring of conditional orthotopic lung cancer in the KP mouse model using an adeno-associated virus vector compatible with biosafety level 1.

BACKGROUND The inducible Kras/p53 lung adenocarcinoma mouse model, which faithfully recapitulates human disease, is routinely initiated by the intratracheal instillation of a virus-based Cre recombinase delivery system. Handling virus-based delivery systems requires elevated biosafety levels, e.g., biosafety level 2 (BSL-2). However, in experimental animal research facilities, following exposure to viral vectors in a BSL-2 environment, rodents may not be reclassified to BSL-1 according to standard practice, preventing access to small animal micro-computed tomography (micro-CT) scanners that are typically housed in general access areas such as BSL-1 rooms. Therefore, our goal was to adapt the protocol so that the Cre-induced KP mouse model could be handled under BSL-1 conditions during the entire procedure. RESULTS The Kras-Lox-STOP-Lox-G12D/p53 flox/flox (KP)-based lung adenocarcinoma mouse model was activated by intratracheal instillation of either an adenoviral-based or a gutless, adeno-associated viral-based Cre delivery system. Tumor growth was monitored over time by micro-CT. We have successfully substituted the virus-based Cre delivery system with a commercially available, gutless, adeno-associated, Cre-expressing vector that allows the KP mouse model to be handled and imaged in a BSL-1 facility. By optimizing the anesthesia protocol and switching to a microscope-guided vector instillation procedure, productivity was increased and procedure-related complications were significantly reduced. In addition, repeated micro-CT analysis of individual animals allowed us to monitor tumor growth longitudinally, dramatically reducing the number of animals required per experiment. Finally, we documented the evolution of tumor volume for different doses, which revealed that individual tumor nodules induced by low-titer AAV-Cre transductions can be monitored over time by micro-CT. CONCLUSION Modifications to the anesthesia and instillation protocols increased the productivity of the original KP protocol. In addition, the switch to a gutless, adeno-associated, Cre-expressing vector allowed longitudinal monitoring of tumor growth under BSL-1 conditions, significantly reducing the number of animals required for an experiment, in line with the 3R principles

Targeting lactate dehydrogenase B-dependent mitochondrial metabolism affects tumor initiating cells and inhibits tumorigenesis of non-small cell lung cancer by inducing mtDNA damage.

Once considered a waste product of anaerobic cellular metabolism, lactate has been identified as a critical regulator of tumorigenesis, maintenance, and progression. The putative primary function of lactate dehydrogenase B (LDHB) is to catalyze the conversion of lactate to pyruvate; however, its role in regulating metabolism during tumorigenesis is largely unknown. To determine whether LDHB plays a pivotal role in tumorigenesis, we performed 2D and 3D in vitro experiments, utilized a conventional xenograft tumor model, and developed a novel genetically engineered mouse model (GEMM) of non-small cell lung cancer (NSCLC), in which we combined an LDHB deletion allele with an inducible model of lung adenocarcinoma driven by the concomitant loss of p53 (also known as Trp53) and expression of oncogenic KRAS (G12D) (KP). Here, we show that epithelial-like, tumor-initiating NSCLC cells feature oxidative phosphorylation (OXPHOS) phenotype that is regulated by LDHB-mediated lactate metabolism. We show that silencing of LDHB induces persistent mitochondrial DNA damage, decreases mitochondrial respiratory complex activity and OXPHOS, resulting in reduced levels of mitochondria-dependent metabolites, e.g., TCA intermediates, amino acids, and nucleotides. Inhibition of LDHB dramatically reduced the survival of tumor-initiating cells and sphere formation in vitro, which can be partially restored by nucleotide supplementation. In addition, LDHB silencing reduced tumor initiation and growth of xenograft tumors. Furthermore, we report for the first time that homozygous deletion of LDHB significantly reduced lung tumorigenesis upon the concomitant loss of Tp53 and expression of oncogenic KRAS without considerably affecting the animal's health status, thereby identifying LDHB as a potential target for NSCLC therapy. In conclusion, our study shows for the first time that LDHB is essential for the maintenance of mitochondrial metabolism, especially nucleotide metabolism, demonstrating that LDHB is crucial for the survival and proliferation of NSCLC tumor-initiating cells and tumorigenesis

Selecting an Appropriate Animal Model of Depression

Depression has become one of the most severe psychiatric disorders and endangers the health of living beings all over the world. In order to explore the molecular mechanism that underlies depression, different kinds of animal models of depression are used in laboratory experiments. However, a credible and reasonable animal model that is capable of imitating the pathologic mechanism of depression in mankind has yet to be found, resulting in a barrier to further investigation of depression. Nevertheless, it is possible to explain the pathologic mechanism of depression to a great extent by a rational modeling method and behavioral testing. This review aims to provide a reference for researchers by comparing the advantages and disadvantages of some common animal depression models

Review of Research on the Three-Dimensional Transition Process of Large-Scale Low-Lift Pump

Due to the uneven distribution of water resources, there are many water diversion projects around the world, such as the South-to-North Water Diversion Project in China, especially in some plain areas. To transfer water from low to high areas, large low-head pumps have been widely used. The transition process of the pumping station is mainly caused by the sudden change in the flow velocity and pressure of the fluid in the pipeline of the pumping station system caused by the start-up and shutdown processes. The previous research has mainly been based on the one-dimensional characteristic line method. However, due to the characteristics of the low-lift pumping station, the flow passage is short and irregular, and the calculation results often cannot guarantee the accuracy of the calculation. In addition to some faults in the actual operation process, in some pumping stations, accidents or operation-scheduling faults are caused by transient processes, such as a high degree of water hammer, the inability to initiate backward flow, the shutdown load rejection runaway exceeding the standard, and decreased hydraulic efficiency. To avoid transition process failures in the newly designed pumping stations and the modified pumping stations, it is necessary to carry out a research review of the three-dimensional transition process of large low-lift pumps. Especially with the development of computing technology, CFD numerical simulation technology has become the main research method for analyzing the pump transition process. The research on the transition process is mainly based on the combination of numerical simulations and experiments. The reliability of a numerical simulation is verified by an experiment. A numerical simulation can measure some parameters that cannot be measured by an experiment. Dynamic mesh technology has become the main technical means for using CFD numerical simulation to study the three-dimensional transition process, and the secondary development of computing software has become the main trend of future development. This paper analyzes and summarizes the research status of the start–stop transition process of large low-lift pump stations and provides a reference for the protection of the start–stop transition process of pump stations

Review of Research on the Three-Dimensional Transition Process of Large-Scale Low-Lift Pump

Due to the uneven distribution of water resources, there are many water diversion projects around the world, such as the South-to-North Water Diversion Project in China, especially in some plain areas. To transfer water from low to high areas, large low-head pumps have been widely used. The transition process of the pumping station is mainly caused by the sudden change in the flow velocity and pressure of the fluid in the pipeline of the pumping station system caused by the start-up and shutdown processes. The previous research has mainly been based on the one-dimensional characteristic line method. However, due to the characteristics of the low-lift pumping station, the flow passage is short and irregular, and the calculation results often cannot guarantee the accuracy of the calculation. In addition to some faults in the actual operation process, in some pumping stations, accidents or operation-scheduling faults are caused by transient processes, such as a high degree of water hammer, the inability to initiate backward flow, the shutdown load rejection runaway exceeding the standard, and decreased hydraulic efficiency. To avoid transition process failures in the newly designed pumping stations and the modified pumping stations, it is necessary to carry out a research review of the three-dimensional transition process of large low-lift pumps. Especially with the development of computing technology, CFD numerical simulation technology has become the main research method for analyzing the pump transition process. The research on the transition process is mainly based on the combination of numerical simulations and experiments. The reliability of a numerical simulation is verified by an experiment. A numerical simulation can measure some parameters that cannot be measured by an experiment. Dynamic mesh technology has become the main technical means for using CFD numerical simulation to study the three-dimensional transition process, and the secondary development of computing software has become the main trend of future development. This paper analyzes and summarizes the research status of the start–stop transition process of large low-lift pump stations and provides a reference for the protection of the start–stop transition process of pump stations

Sediment Erosion on Pelton Turbines: A Review

Abstract The Pelton turbine has been widely used to develop high-head water resources with sediments because of its advantages in life cycle costs. When a flood or monsoon season occurs, the sediment concentration in the river increases suddenly, causing severe erosion to the nozzle, needle, and runner of Pelton turbines. After decades of development, researchers have developed practical engineering experience to reduce the sediment concentration of the flow through the turbine and ensure the safety and efficiency of power generation. Research on the mechanism of sediment erosion, development of anti-erosion materials, and establishment of erosion prediction models have attracted scholarly interest in recent years. Extensive research has been conducted to determine a complete and valuable syndication erosion model. However, owing to the complexity of the flow and wear mechanisms, the influence of specific parameters of erosion and the syndication effect is still difficult to determine. Computational fluid dynamics and erosion monitoring technology have also been evaluated and applied. This paper presents a comprehensive review of the erosion of Pelton turbines, some of the latest technical methods, and possible future development directions

SF3B3-regulated mTOR alternative splicing promotes colorectal cancer progression and metastasis

Abstract Background Aberrant alternative splicing (AS) is a pervasive event during colorectal cancer (CRC) development. SF3B3 is a splicing factor component of U2 small nuclear ribonucleoproteins which are crucial for early stages of spliceosome assembly. The role of SF3B3 in CRC remains unknown. Methods SF3B3 expression in human CRCs was analyzed using publicly available CRC datasets, immunohistochemistry, qRT-PCR, and western blot. RNA-seq, RNA immunoprecipitation, and lipidomics were performed in SF3B3 knockdown or overexpressing CRC cell lines. CRC cell xenografts, patient-derived xenografts, patient-derived organoids, and orthotopic metastasis mouse models were utilized to determine the in vivo role of SF3B3 in CRC progression and metastasis. Results SF3B3 was upregulated in CRC samples and associated with poor survival. Inhibition of SF3B3 by RNA silencing suppressed the proliferation and metastasis of CRC cells in vitro and in vivo, characterized by mitochondria injury, increased reactive oxygen species (ROS), and apoptosis. Mechanistically, silencing of SF3B3 increased mTOR exon-skipped splicing, leading to the suppression of lipogenesis via mTOR-SREBF1-FASN signaling. The combination of SF3B3 shRNAs and mTOR inhibitors showed synergistic antitumor activity in patient-derived CRC organoids and xenografts. Importantly, we identified SF3B3 as a critical regulator of mTOR splicing and autophagy in multiple cancers. Conclusions Our findings revealed that SF3B3 promoted CRC progression and metastasis by regulating mTOR alternative splicing and SREBF1-FASN-mediated lipogenesis, providing strong evidence to support SF3B3 as a druggable target for CRC therapy