Similar gut bacteria composition in Apriona germari on two preferred host plants.

Apriona germari is one of the most serious wood-boring pests that cause damage to economic and landscaping trees and has adapted to a wide range of plants as diet. Gut bacteria play an important role in biology and ecology of herbivores, especially in growth and adaptation. To investigate how plant hosts shape A. germari gut microbiota, A. germari larvae were collected from Populus tomentosa and Malus pumilal, and gut microbiomes were sequenced based on 16S rDNA high-throughput sequencing technology. A total of 853,424 high-quality reads were obtained and clustered into 196 operational taxonomic units under a 97% similarity cutoff, which were annotated into 8 phyla, 10 classes, 21 orders, 34 families, 59 genera, and 39 species. Gibbsiella was the most dominant genus of intestinal bacteria, followed by Enterobacter and Acinetobacter. No significant difference was observed in larvae gut bacterial richness and diversity of A. germari collected from two hosts, though alpha diversity showed that the richness of gut bacteria in A. germari larvae collected on P. tomentosa was slightly higher than that in A. germari on M. pumilal, and beta diversity showed little difference between two host plants. The functional abundance analysis of the detected bacteria revealed fermentation, chemoheterotrophy, symbionts, and nitrate relative functions that highly possibly support wood-boring beetles to feed on woody tissues. Our study provided a theoretical basis for investigating the function of intestinal symbiosis bacteria of A. germari.

Low Expression of Pseudogene POU5F1B Affects Diagnosis and Prognosis in Acute Myeloid Leukemia (AML).

BACKGROUND The transcription factor Oct-4 is necessary for maintaining pluripotency and self-renewal of embryonic stem cells, and POU5F1B is a processed pseudogene of Oct-4 with coding capacity. The purpose of this study is to evaluate the expression and clinical implication of POU5F1B in AML. MATERIAL AND METHODS The expression of the POU5F1B transcript was evaluated in 175 newly diagnosed AML patients and 39 healthy controls by use of real-time quantitative PCR (RQ-PCR). RESULTS POU5F1B was underexpressed in AML compared with controls (P<0.001). The receiver operating characteristic (ROC) curve revealed that the POU5F1B transcript level was able to differentiate AML patients from healthy individuals (AUC=0.682). In non-APL AML patients, the POU5F1Blow group had significantly higher WBC than the POU5F1Bhigh group (20.2×109 vs. 4.6×109 L⁻¹, P=0.021). Among whole-cohort AML, non-APL AML, and intermediate-risk AML, POU5F1Bhigh patients had obviously higher complete remission (CR) rates than POU5F1Blow patients (P=0.012, P=0.012 and P=0.027). In addition, Kaplan-Meier analysis demonstrated better overall survival (OS, P=0.019, P=0.007 and P=0.046, respectively) in POU5F1Bhigh patients compared with POU5F1Blow patients. Furthermore, in multivariate survival analysis, POU5F1B was independently associated with OS in non-APL AML patients and intermediate-risk AML as a favorable prognostic factor. CONCLUSIONS POU5F1B was frequently underexpressed in AML, and might contribute to the diagnosis and prognosis of AML.

[Spatial-temporal Variation in Water Quality of Rain-source Rivers in Shenzhen from 2015 to 2021 and Its Response to Rainfall].

Rain-source urban rivers have the characteristics of small water capacity， lack of dynamic water supply， and being easily polluted. This study analyzed the spatial and temporal distribution characteristics of river water quality and the response of characteristic pollutants to rainfall based on daily rainfall data and 21 water quality indicators of nine major river basins in Shenzhen （excluding Shenzhen-Shantou） from 2015 to 2021 by using the single-factor assessment method， comprehensive pollution index method， hierarchical cluster analysis， and Pearson correlation. The results showed that： ① in 2015， the water quality of most sections in the whole region was inferior Class V water. After October 2018， the overall water quality of rivers was greatly improved， which was consistent with the background of Shenzhen's special water control activities in 2018. By 2021， the water quality of approximately 62% of sections reached Class Ⅰ-Ⅲ water standards. ② The water pollution in the densely populated western part of Shenzhen was more serious than that in the eastern part， and the water pollution in the lower reaches of the estuaries and tributaries was more serious than that in the upper reaches. ③ The water quality of the Pingshan River， Guanlan River， Longgang River， and Maozhou River was significantly affected by rainfall. ④ The main characteristic pollution indexes of the Shenzhen River were DO， permanganate index， COD， BOD5， NH4+-N， TP， petroleum， and anionic surfactant. For the Pingshan River and Longgang River， rainfall increased the concentrations of TP and NH4+-N. For the Maozhou River， rainfall increased the concentrations of TP and COD. For the Shenzhen River， rainfall increased the concentrations of COD， TP， and NH4+-N. The above results reveal the spatio-temporal variation in rain-source river water quality in Shenzhen and its response to non-point source pollution caused by rainfall events and provide a scientific reference for building a higher quality water environment in Shenzhen.

Circular RNA BMI1 Serves as a Potential Target for Diagnosis and Treatment in Esophageal Cancer.

AIMS: Previous studies have confirmed that BMI1 is elevated in esophageal cancer, which is a potential therapeutic target for esophageal cancer. However, the clinical significance of circular RNA BMI1 (circ-BMI1) in esophageal cancer is not yet clear. Herein, we revealed the clinical implication of circ-BMI1 in esophageal cancer, and provided a theoretical basis for molecular diagnosis and potential targeted therapy of esophageal cancer. METHODS: Firstly, 10 fresh paired esophageal cancer tissues and paracancer tissues, 49 esophageal cancer serum samples and 28 healthy control serum samples were involved in our study. Differential expression and clinical significance of circ-BMI1 in esophageal cancer patients and healthy controls were evaluated by quantitative Real-time RT-PCR (RT-qPCR). Secondly, effects of circ-BMI1 differential expression on biological function of esophageal cancer cell line Eca109 were analyzed. Effects of circ-BMI1 on cell proliferation, migration and colony forming ability were evaluated by CCK-8, wound healing, and colony-forming assay. Cell apoptosis, drug sensitivity tests were also be conducted. Finally, influence of Eca109 cells differentially expressed by circ-BMI1 on tumorigenicity in nude mice was studied. RESULTS: Expression of circ-BMI1 in serum and tissues of esophageal cancer patients was significantly decreased compared to controls (P < 0.001 and P = 0.003, respectively). Area under the receiver operating characteristic curve (ROC) was 0.726. Cell proliferation, migration and colony forming ability of circBMI1-Eca109 cells were obviously decreased than that of NC-Eca109 cells (P < 0.05). circBMI1-Eca109 cells were more sensitive to 5-fluorouracil and cisplatin, and tumor volume of nude mice in circBMI1-Eca109 group was smaller (P < 0.05). CONCLUSIONS: The study indicated that expression of circ-BMI1 was significantly down-regulated in esophageal cancer. Overexpression of circ-BMI1 inhibited proliferation, migration, colony formation of Eca109 cells, and tumor growth of Eca109 cells in nude mice. circ-BMI1 may be a potential target for diagnosis and treatment in esophageal cancer.

Circ_0002232 Acts as a Potential Biomarker for AML and Reveals a Potential ceRNA Network of Circ_0002232/miR-92a-3p/PTEN.

PURPOSE: Our research aimed to investigate the expression level of circ_0002232, which is transcribed from PTEN, and find out the association of circ_0002232/miR-92a-3p/PTEN network in acute myeloid leukemia (AML). METHODS: Circ_0002232 expression in 115 AML patients and 48 controls was detected by using real-time quantitative PCR. The diagnostic value of circ_0002232 expression was evaluated by receiver operating characteristic curve. Kaplan-Meier curves were used to analyse the impact of circ_0002232 for overall survival. Associated network of circ_0002232 was predicted by using interaction prediction websites. RESULTS: Compared with controls, circ_0002232 was notably low-expressed in AML (P<0.001). According to the result of receiver operating characteristic curve, circ_0002232 expression could distinguish AML patients from controls (P<0.001). There were significant differences in patients' age (P=0.004), FAB classifications (P=0.036), white blood cell count (P=0.041) and platelet count (P=0.021) between low-expressed circ_0002232 group and high-expressed circ_0002232 group. Moreover, there was a positive correlation between circ_0002232 expression and patients' age (Pearson r=0.256, P=0.0057). Interestingly, we found that patients in low-expressed circ_0002232 group had better overall survival both in whole AML (P=0.030) and non-APL AML (P=0.014). Remarkably, the expression of circ_0002232 was positively correlated with PTEN (Spearman r=0.678, P<0.001). Furthermore, there was a negative correlation in AML between circ_0002232 and miR-92a-3p (Spearman r=-0.301, P=0.016), miR-92a-3p and PTEN (Spearman r=-0.324, P=0.034). Interaction prediction websites revealed that circ_0002232 might affect the expression of PTEN and the process of AML through sponging miR-92a-3p. CONCLUSION: Circ_0002232, one of the circRNAs transcribed from PTEN, was remarkably down-regulated in AML and could act as a promising biomarker for the diagnosis of AML. In addition, there might be a potential association network of circ_0002232/miR-92a-3p/PTEN in AML.

Polysaccharides extracted from the roots of Bupleurum chinense DC modulates macrophage functions.

The present study aimed to investigate the effects of polysaccharides extracted from Bupleurum chinense DC (BCPs) on macrophage functions. In the in vivo experiment, 1 mL of 5% sodium thioglycollate was injected into the abdomen of the mice on Day 0 and macrophages were harvested on Day 4. The macrophages were cultured in plates and treated with different concentrations of BCPs and stimulus. Effects of BCPs on macrophage functions were assessed by chemotaxis assay, phagocytosis assay and Enzyme-Linked Immunosorbent Assay (ELISA). Our results showed the enhanced chemotaxis, phagocytosis and secretion of nitric oxide (NO) and inflammatory cytokines by macrophages when treated with BCPs. However, when chemotaxis and phagocytosis were up-regulated by complement components or opsonized particles, BCPs inhibited these effects. Also, the NO production induced by lipopolysaccharides (LPS) was suppressed by BCPs mildly. Moreover, BCPs had an inhibitory effect on the [Ca2+]i elevation of macrophages. These results suggested that BCPs exerted modulatory effects on macrophage functions, which may contribute to developing novel approaches to treating inflammatory diseases.

[Study of signal transduction mediated by integrin alphaIIbbeta3 using a dominant negative model].

This study was purposed to investigate the role of integrin beta3 cytoplasmic domain in signal transduction mediated by integrin alphaIIbbeta3 and to explore the effect of integrin beta3 on signal transduction and specificity in condition without alphaIIb subunit. The fusion protein (Tac/beta3) was stably expressed in CHO cell line expressing GPIbIX, integrin alphaIIbbeta3 (IbIX/IIbIIIa-CHO cell line) by combining extracellular and transmembrane domains (Tac) of IL-2 receptor with integrin beta3 cytoplasmic domain (beta3) for formation of fusion protein (Tac/beta3). Then a series of tests were performed, including spreading and stable adhesion of IbIX/IIbIIIa-CHO cell line in solid phase fibrinogen (Fg), fibrin clot restriction and soluble fibrinogen binding, which represent outside-in and inside-out signal transduction events. The results showed that the bidirectional signal transduction mediated by alphaIIbbeta3 in IbIX/IIbIIIa-CHO/Tac-762 cells stably expressing Tac/beta3 was seriously inhibited. It is concluded that the Tac/beta3 can play a significant role in IbIX/IIbIIIa-CHO/Tac-762 cells through a dominant negative mode, the independent presence of beta3 subunit cytoplasmic domain can regulate the bidirectional signal transduction mediated by integrin alphaIIbbeta3.

[Preparation and characterization of a monoclonal antibody against human c-Kit].

AIM: To prepare anti-c-Kit monoclonal antibodies and characterize their specificity of epitope recognition. METHODS: cDNA encoding human c-Kit extracellular domain was constructed into a procaryotic expression vector pQE30 and the correctness of the reconstructed plasmid pQE30-KitD4-5 was verified by sequencing. The plasmid was transformed into E.coli M15 strain. Recombinant 6 x His pQE30-KitD4-5 was expressed after induction by IPTG for 4 h. Then SDS-PAGE results suggested that the products mainly formed inclusion bodies. The fusion protein was further purified with Ni-NTA-His affinity chromatography and then used to immunize BALB/c mice. The hybridomas were achieved by fusing the immunized spleen cells with the Sp2/0 myeloma cell line. The positive clones were screened by FCM with CHO-hKit cells. Hybidoma clones secreting anti-c-Kit antibodies were further subcloned and investigated for their biological activities by Western blot, rapid isotyping analysis and FCM. RESULTS: Recombinant human c-Kit fusion proteins were in vitro expressed and purified to be used as immunogen. One stable hybridoma cell line, which continuously secrets specific anti-c-Kit monoclonal antibody ((SRJ1)) was established. The biological activity studies showed that the monoclonal antibody recognized the natural c-Kit expressed on the Kasumi leukemia cell line, but failed to bind to the normal human peripheral blood cells. Interestingly, this monoclonal antibody failed to recognize a subpopulation of Kasumi cells that is reactive with the commercial anti-c-Kit mAb Ab81 suggesting that the c-Kit expressed by this subpopulation contains some sequencial and/or structural aberrations that are distinguishable by mAb SRJ1. CONCLUSION: With an immunization procedure using purified recombinant human c-Kit fusion proteins. a hybridoma cell line continuously and stably secreting anti-c-Kit monoclonal antibody has been established. The monoclonal antibody SRJ1 specifically recognizes human c-Kit expressed on the leukemia cells, and may provide a novel approach to analyze the possible structural variations of c-Kit expressed by different cells.

Fibrinogen interaction of CHO cells expressing chimeric alphaIIb/alphavbeta3 integrin.

AIM: The molecular mechanisms of the affinity regulation of alphavbeta3 integrin are important in tumor development, wound repairing, and angiogenesis. It has been established that the cytoplasmic domains of alphavbeta3 integrin play an important role in integrin-ligand affinity regulation. However, the relationship of structure-function within these domains remains unclear. METHODS: The extracellular and transmembrane domain of alphaIIb was fused to the alphav integrin cytoplasmic domain, and the chimeric alpha subunit was coexpressed in Chinese hamster ovary (CHO) cells with the wild-type beta3 subunit or with 3 mutant beta3 sequences bearing truncations at the positions of T741, Y747, and F754, respectively. The CHO cells expressing these recombinant integrins were tested for soluble fibrinogen binding and the cell adhesion and spreading on immobilized fibrinogen. RESULTS: All 4 types of integrins bound soluble fibrinogen in the absence of agonist stimulation, and only the cells expressing the chimeric alpha subunit with the wild-type beta3 subunit, but not those with truncated beta3, could adhere to and spread on immobilized fibrinogen. CONCLUSION: The substitution alphaIIb at the cytoplasmic domain with the alphav cytoplasmic sequence rendered the extracellular alphaIIbbeta3 a constitutively activated conformation for ligands without the need of pinside-outq signals. Our results also indicated that the COOH-terminal sequence of beta3 might play a key role in integrin alphaIIb/alphavbeta3-mediated cell adhesion and spreading on immobilized fibrinogen. The cells expressing alphaIIb/alphavbeta3 have enormous potential for facilitating drug screening for antagonists either to alphavbeta3 intracellular interactions or to alphaIIbbeta3 receptor functions.