CRISPR/Cas9-Bibliothek identifiziert PIP4K2C als Regulator von PVR

1.1 Background and objectives: Malignant tumors of the breast are common among women, and they represent the number one threat to their health, especially triple-negative breast cancer. It is a relatively common cause of death for women. At present, immunotherapy has become an emerging method of cancer treatment. Cancer cells express increased levels of PVR, which is classified as a cell adhesion molecule. The molecule is expressed in a variety of tumor cells, including breast cancer, pancreatic cancer, gallbladder cancer, liver cancer, ovarian cancer, etc. In the development of tumors, it is involved in the adhesion, metastasis and proliferation of tumors. A PVR-mediated tumor growth mechanism does not just affect the growth of the tumor, but also influences the tumor immune environment, and takes part in immune escape to some extent. However, the expression of PVR is not limited to the area where immune cells exist and is uniformly expressed in tumor cells. Therefore, the expression and regulation of PVR should not be affected by external factors but caused by certain gene mutations in the tumor cells. In addition, PVR expression is related to the tumor's development stage. Early and late stages of tumor development are markedly different in their expression of PVR, and its role also differs. Additionally, PVR expression is closely related to that of PD-L1, a tumor immune target for which tumor immunotherapy is now more concerned, and this also affects the efficacy of tumor immunotherapy. As a result, the biomarker PVR was chosen for the purposes of this research. 1.2 Methods: The aim of the study is to identify the targets that regulate PVR in breast cancer using CRISPR/Cas9 loss-of-function screening technology, as well as to strengthen the screening through fluorescence-activated cell sorting (FACS) to identify groups expressing high and low PVR expression levels. We transfected breast cancer cell line with the human protein kinase library. After three rounds of FACS cell sorting, the cell population with high and low expression of PVR was detected by flow cytometry and Western Blot, and the DNA with 500-fold coverage was collected for next-generation sequencing to identify the candidate gene. 1.3 Results: In this study, we analyzed PIP4K2C as an important gene regulating PVR expression, and knocking it down resulted in increased expression of PVR in cells. Furthermore, we demonstrate that this regulation involves the AKT/mTOR signaling pathway and is found to promote cancer cell migration in RAS-mutated cells. 1.4 Conclusion: Our results support the utility and effectiveness of using CRISPR/Cas9 combined FACS technology to screen immune targets of interest and provide new targets to regulate PVR expression. PIP4K2C also plays an important role in regulating the expression of PVR, and AKT/mTOR is its potential signaling pathway

Expression, Purification Strategy and Detection Method Establishment of Anti-Fenitrothion Nanobody

For the purpose of preparing anti-fenitrothion nanobodies with high-purity efficiently, the expression conditions of a recombinant anti-fenitrothion nanobody with low expression level in E. coli was optimized. The IPTG concentration and inducing temperature were selected as the independent variables and the nanobody expression level was used as the dependent variable for single-factor experiments, and the purification strategy was also investigated. The results showed that the highest expression level of 6 mg/L anti-fenitrothion nanobody was achieved at 37 ℃ without IPTG. Moreover, an interactive effect of IPTG dosage and inducing temperature was found. After a two-step purification of Ni affinity chromatography and gel filtration chromatography, the anti-fenitrothion nanobodies was finally yielded with more than 98% purity, indicating that the expression and purification strategies in this study can obtain anti-fenitrothion nanobodies efficiently. An ic-ELISA assay was then established based on the obtained nanobody, with an IC50 of 5.81 ng/mL and an LOD of 0.25 ng/mL, and a detection range of 0.78~43.07 ng/mL. The assay was applied to determine fenitrothion in Chinese cabbage and lettuce samples. The recovery rate of addition was between 93.3%~111.7%, and the coefficient of variation (CV) was between 2.3%~18.2%. The proposed assay based on anti-fenitrothion nanobody has high sensitivity, which can meet the requirements for monitoring of fenitrothion under the national standard, thus can be used for rapid screening of fenitrothion. This study laid a foundation for the development of efficient and rapid immunoassays for fenitrothion

Filamin A Is a Potential Driver of Breast Cancer Metastasis via Regulation of MMP-1

Recurrent metastasis is a major fatal cause of breast cancer. Regretfully, the driving force and the molecular beneath have not been fully illustrated yet. In this study, a cohort of breast cancer patients with locoregional metastasis was recruited. For them, we collected the matched samples of the primary tumor and metastatic tumor, and then we determined the mutation profiles with whole-exome sequencing (WES). On basis of the profiles, we identified a list of deleterious variants in eight susceptible genes. Of them, filamin A (FLNA) was considered a potential driver gene of metastasis, and its low expression could enhance 5 years’ relapse survival rate by 15%. To prove the finding, we constructed a stable FLNA knockout tumor cell line, which manifested that the cell abilities of proliferation, migration, and invasion were significantly weakened in response to the gene knockout. Subsequently, xenograft mouse experiments further proved that FLNA knockout could inhibit local or distal metastasis. Putting all the results together, we consolidated that FLNA could be a potential driver gene to metastasis of breast cancer, in particular triple-negative breast cancer. Additional experiments also suggested that FLNA might intervene in metastasis via the regulation of MMP-1 expression. In summary, this study demonstrates that FLNA may play as a positive regulator in cancer proliferation and recurrence. It provides new insight into breast cancer metastasis and suggests a potential new therapeutic target for breast cancer therapy

Habitat Use and Activity Patterns of Mammals and Birds in Relation to Temperature and Vegetation Cover in the Alpine Ecosystem of Southwestern China with Camera-Trapping Monitoring

The high-altitude ecosystem of the Tibetan Plateau in China is a biodiversity hotspot that provides unique habitats for endemic and relict species along an altitudinal gradient at the eastern edge. Acquiring biodiversity information in this area, where the average altitude is over 4000 m, has been difficult but has been aided by recent developments in non-invasive technology, including infrared-triggered camera trapping. We used camera trapping to acquire a substantial number of photographic wildlife records in Wolong National Nature Reserve, Sichuan, China, from 2013 to 2016. We collected information of the habitat surrounding the observation sites, resulting in a dataset covering 37 species and 12 environmental factors. We performed a multivariate statistical analysis to discern the dominant environmental factors and cluster the mammals and birds of the ecosystem in order to examine environmental factors contributing to the species’ relative abundance. Species were generalized into three main types, i.e., cold-resistant, phyllophilic, and thermophilic, according to the identified key environmental drivers (i.e., temperature and vegetation) for their abundances. The mammal species with the highest relative abundance were bharal (Pseudois nayaur), Moupin pika (Ochotona thibetana), and Himalayan marmot (Marmota himalayana). The bird species with highest relative abundance were snow partridge (Lerwa lerwa), plain mountain finch (Leucosticte nemoricola), Chinese monal (Lophophorus lhuysii), and alpine accentor (Prunella collaris)

Global behavior of a multi-group SEIR epidemic model with spatial diffusion in a heterogeneous environment

In this paper, we propose a multi-group SEIR epidemic model with spatial diffusion, where the model parameters are spatially heterogeneous. The positivity and ultimate boundedness of the solution, as well as the existence of a global attractor of the associated solution semiflow, are established. The definition of the basic reproduction number is given by utilizing the next generation operator approach, whereby threshold-type results on the global dynamics in terms of this number are established. That is, when the basic reproduction number is less than one, the disease-free steady state is globally asymptotically stable, while if it is greater than one, uniform persistence of this model is proved. Finally, the feasibility of the main theoretical results is shown with the aid of numerical examples for a model with two groups

A space-for-time (SFT) substitution approach to studying historical phenological changes in urban environment.

Plant phenological records are crucial for predicting plant responses to global warming. However, many historical records are either short or replete with data gaps, which pose limitations and may lead to erroneous conclusions about the direction and magnitude of change. In addition to uninterrupted monitoring, missing observations may be substituted via modeling, experimentation, or gradient analysis. Here we have developed a space-for-time (SFT) substitution method that uses spatial phenology and temperature data to fill gaps in historical records. To do this, we combined historical data for several tree species from a single location with spatial data for the same species and used linear regression and Analysis of Covariance (ANCOVA) to build complementary spring phenology models and assess improvements achieved by the approach. SFT substitution allowed increasing the sample size and developing more robust phenology models for some of the species studied. Testing models with reduced historical data size revealed thresholds at which SFT improved historical trend estimation. We conclude that under certain circumstances both the robustness of models and accuracy of phenological trends can be enhanced although some limitations and assumptions still need to be resolved. There is considerable potential for exploring SFT analyses in phenology studies, especially those conducted in urban environments and those dealing with non-linearities in phenology modeling

Destruction of the Dorsal Motor Nucleus of the Vagus Aggravates Inflammation and Injury from Acid-Induced Acute Esophagitis in a Rat Model

Background/Aims. The aim of this study is to examine the protective effect of the cholinergic anti-inflammatory pathway (CAP) in experimental esophagitis in rats. Methods. A total of 40 male Sprague-Dawley (SD) rats were randomly divided into five groups as follows: control group, sham + saline group, sham + acid group, operation + saline group, and operation + acid group. Two weeks after the dorsal motor nucleus of the vagus (DMV) destruction, hydrochloric acid with pepsin was perfused into the lower part of the esophagus for 90 min. The rats were sacrificed 60 min after perfusion. The esophagus was prepared for hematoxylin and eosin (HE) staining, and the degree of inflammation and NF-κB activation in the esophagus was measured. Inflammatory cytokines (TNF-α, IL-6, IL-1β, and PGE2) in the esophagus were measured by ELISA. The brain was removed and processed for c-fos immunohistochemistry staining. The c-fos-positive neurons were counted and analyzed. Results. The TNF-α, IL-1β, IL-6, and PGE2 concentrations in the esophageal tissue increased after acid perfusion. The microscopic esophagitis scores and the activation of NF-κB p65 in the esophagus were significantly higher in the operation + acid group than in the operation + saline group. c-fos-positive neurons significantly increased in rats receiving acid perfusion in the amygdala (AM), the paraventricular nucleus of the hypothalamus (PVN), the parabrachial nucleus (PBN), the nucleus of the solitary tract (NTS)/DMV, the nucleus ambiguous (NA), the reticular nucleus of the medulla (RNM), and the area postrema (AP). After DMV destruction, c-fos expression was reduced in the AM, PVN, PBN, NTS/DMV, NA, RNM, and AP, especially in the AM, PVN, NTS/DMV, RNM, and AP. Conclusions. The DMV is an important nucleus of the CAP. The DMV lesion can aggravate esophageal inflammation and injury from acid-induced acute esophagitis in a rat model. The CAP has a protective effect on the acute esophagitis rat model and could be a new therapy for reflux esophagitis (RE)

Cold water interception cooling technology for surrounding rock of heat-damaged mine

In order to alleviate the problem of mine thermal damage, this paper proposes to put cold water pipe into the surrounding rock of roadway, and reduce the temperature of surrounding rock by intercepting the high temperature through the cold water pipe. According to the similarity principle, a cold water interception and cooling experimental system for high temperature surrounding rock was built. Experiments were conducted by changing cold water velocity, inlet time and surrounding rock temperature, etc. Combined with COMSOL simulation, the influence of different diameters and spacing layouts of cold water pipe on cold water interception and cooling effect of roadway surrounding rock was analyzed. The results show that when other conditions remain unchanged, the cooling range of surrounding rock increases with the increase of cold water velocity, inlet time and temperature difference between surrounding rock and cold water. When the diameter of the cold water pipe is 10 cm and the vertical distance is 2.5 m, the average temperature of the roadway wall after cooling is 23.4 ℃

Measurement and distribution of surrounding rock cooling zone in Yangchangwan Coal Mine

In this paper, the deep hole and shallow hole temperature measurement technology is used to measure the rock temperature in No.13 mining area of Yangchangwan Coal Mine, and the cooling circle range of 937 m elevation roadway for the return air downhill in No.13 mining area and 430 m elevation roadway for the auxiliary transportation downhill in No.13 mining area are determined. The COMSOL software is used to analyze the surrounding rock temperature distribution, and the reliability of the surface borehole temperature measurement data is verified according to the temperature measurement results. Combined with the ground borehole temperature measurement data and the deep hole and shallow hole temperature measurement results, the ground temperature gradient and the heat damage area are defined, which provides a basis for the subsequent heat damage treatment

Genome-Wide Identification and Characterization of Banana Ca2+-ATPase Genes and Expression Analysis under Different Concentrations of Ca2+ Treatments

Ca2+-ATPases have been confirmed to play very important roles in plant growth and development and in stress responses. However, studies on banana (Musa acuminata) Ca2+-ATPases are very limited. In this study, we identified 18 Ca2+-ATPase genes from banana, including 6 P-IIA or ER (Endoplasmic Reticulum) type Ca2+-ATPases (MaEACs) and 12 P-IIB or Auto-Inhibited Ca2+-ATPases (MaACAs). The MaEACs and MaACAs could be further classified into two and three subfamilies, respectively. This classification is well supported by their gene structures, which are encoded by protein motif distributions. The banana Ca2+-ATPases were all predicted to be plasma membrane-located. The promoter regions of banana Ca2+-ATPases contain many cis-acting elements and transcription factor binding sites (TFBS). A gene expression analysis showed that banana Ca2+-ATPases were differentially expressed in different organs. By investigating their expression patterns in banana roots under different concentrations of Ca2+ treatments, we found that most banana Ca2+-ATPase members were highly expressed under 4 mM and 2 mM Ca2+ treatments, but their expression decreased under 1 mM and 0 mM Ca2+ treatments, suggesting that their downregulation might be closely related to reduced Ca accumulation and retarded growth under low Ca2+ and Ca2+ deficiency conditions. Our study will contribute to the understanding of the roles of Ca2+-ATPases in banana growth and Ca management