[Effects of Nintedanib associated with Shenfu Injection on paraquat-induced lung injury in rats].

Objective: To study the effects of Nintedanib associated with Shenfu Injection on lung injury induced by paraquat (PQ) intoxication. Methods: In September 2021, a total of 90 SD rats were divided into 5 groups in random, namely control group, PQ poisoning group, Shenfu Injection group, Nintedanib group and associated group, 18 rats in each group. Normal saline was given by gavage route to rats of control group, 20% PQ (80 mg/kg) was administered by gavage route to rats of other four groups. 6 hours after PQ gavage, Shenfu Injection group (12 ml/kg Shenfu Injection), Nintedanib group (60 mg/kg Nintedanib) and associated group (12 ml/kg Shenfu Injection and 60 mg/kg Nintedanib) were administered with medicine once a day. The levels of serum transforming growth factor beta1 (TGF-ß1), interleukin-1 beta (IL-1ß) were determined at 1, 3 and 7 d, respectively. The pathological changes of lung tissue, the ratio of wet weight and dry weight (W/D) of lung tissue, the levels of superoxide dismutase (SOD) and malondialdehyde (MDA) in lung tissue were observed and determined after 7 d. Western blot was used to analyse the expression levels of fibroblast growth factor receptor 1 (FGFR1), platelet derivation growth factor receptor alpha (PDGFRα), vascular endothelial growth factor receptor 2 (VEGFR2) in lung tissue after 7 d. Results: The levels of TGF-ß1, IL-1ß in all poisoning groups went up first and then went down. The levels of TGF-ß1, IL-1ß in associated group at 1, 3, 7 d were lower than that of PQ poisoning group, Shenfu Injection group and Nintedanib group at the same point (P<0.05). Pathological changes of lung tissue under the light microscopes showed that the degrees of hemorrhage, effusion and infiltration of inflammatory cells inside the alveolar space of Shenfu Injection group, Nintedanib group and associated group were milder than that of PQ poisoning group, and the midest in associated group. Compared with control group, the W/D of lung tissue was higher, the level of MDA in lung tissue was higher, while the level of SOD was lower, the expressions of FGFR1, PDGFRα and VEGFR2 in lung tissue were higher in PQ poisoning group (P<0.05). Compared with PQ poisoning group, Shenfu Injection group and Nintedanib group, the W/D of lung tissue was lower, the level of MDA in lung tissue was lower, while the level of SOD was higher, the expressions of FGFR1, PDGFRα and VEGFR2 in lung tissue were lower in associated group (P<0.05) . Conclusion: Nintedanib associated with Shenfu Injection can relieve lung injury of rats induced by PQ, which may be related to Nintedanib associated with Shenfu Injection can inhibit the activation of TGF-ß1 and the expressions of FGFR1, PDGFRα, VEGFR2 in lung tissue of rats.

[Clinicopathological characteristics and prognosis of diffuse midline gliomas with histone H3K27M mutation: an analysis of 30 cases].

Objective: To analyze the clinicopathological characteristics and prognosis of diffuse midline glioma (DMG) with H3K27M mutation. Methods: Thirty cases of DMG were collected in Guangdong Sanjiu Brain Hospital from October 2016 to May 2018. The patients' clinicopathological data including age, tumor site and histological grade, treatment and follow-up data were collected and analyzed. Results: There were 21 males and 9 females, with a mean age of 26 years (range 5-53 years). Fourteen tumors were located in thalamus, 12 in brainstem (one involved both thalamus and brainstem), and one each in hypothalamus, fourth ventricle, and sellar region, respectively. Two cases presented as diffuse intracranial lesions. Three cases (10.0%) were of WHO grade Ⅰ, 10 cases (33.3%) were grade Ⅱ, eight cases (26.7%) were grade Ⅲ, and nine cases (30.0%) were grade Ⅳ.All patients with gradeⅠ tumors were older than 20 years. Histologically, all were pilocytic astrocytoma-like. Immunohistochemical staining demonstrated that all tumors were IDH1 negative. Twenty-eight tumors showed diffuse expression of H3K27M, and two showed focal expression. Twenty-one tumors(100.0%, 21/21) showed absent expression of H3K27me3. Sixteen tumors (57.1%, 16/28) showed strongly positive expression of p53, and ATRX was negative in eight tumors (38.1%, 8/21). The Ki-67 proliferation index ranged from 5% to 40%. Eight cases (including two cases of H3K27M expression of individual cells) showed K27M mutation in H3F3A gene. Intracranial and spinal cord dissemination occurred in six cases (20.0%, 6/30). Median progression-free survival (PFS) was 9.5 months and median overall survival (OS) was 34 months. Mean PFS was 11.2 months and mean OS was 24.3 months. Compared with adults (>20 years old), children/adolescents (no more than 20 years old) had significantly shorter median OS (8 months vs. 34 months, P=0.013). There was no significant difference in PFS and OS between DMGs located in the brain stem/thalamus and other sites within midline (P>0.05). There was no significant difference in PFS and OS between WHO grade ⅠDMGs and WHO grade Ⅱ-Ⅳ DMGs (P>0.05). Conclusions: DMGs occur more commonly in children and adolescents with male predominance. DMGs present with WHO Ⅰ-Ⅳ tumors morphologically, and pilocytic astrocytoma-like lesions with WHO Ⅰ are more common in adults. Expression of H3K27M but not H3K27me3 is helpful for diagnosis of DMG. The prognosis of children/adolescents is significantly worse than that of adults, whereas histological grade and tumor location do not affect prognosis.

[Effects of neutrophils alkaline phosphatase on functions of neutrophils in vitro].

OBJECTIVE: To investigate the effects of neutrophils alkaline phosphatase (NAP) on the migration, reactive oxygen species (ROS) generation and apoptosis of neutrophil-like differentiated HL-60 cells. METHODS: NAP was overexpressed in HL-60 cells via transfecting coding sequence of NAP by lentivirus. The effectivity of NAP overexpression was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. HL-60 cells were differentiated into neutrophil-like cells by exposure to 1.5% DMSO. The migration and ROS generation of neutrophil-like cells with NAP overexpression were detected by transwell migration test and flow cytometry, respectively. Cell apoptosis were detected by flow cytometry. The expression of apoptosis-related protein Bax, caspase-3 and caspase-9 in neutrophil-like cells were observed by Western blotting after NAP overexpression. RESULTS: Over 80% HL-60 cells presented green fluorescence after GFP-NAP infection by lentivirus and screening by puromycin. In addition, the levels of both gene and protein of NAP were up-regulated in these cells. After 5-day culture with 1.5% DMSO, the bulk of induced cell was smaller and the surface appeared many bumps and irregular shape. The ratio of nuclear and cytoplasmic decreased and nucleolus disappeared. The nuclear chromatin changed from dense to loose and the nuclear morphology appeared to be rod and segmented. The percentage of CD11b(+) cell increased from 26.25% to 98.55%. The transwell migration test showed that the number of migrated cells was higher in neutrophil-like cells with NAP overexpression compared with the negative control [(15.30±3.65) ×10(3) vs (8.00±0.78) ×10(3)] (P<0.001). RESULTS of flow cytometry suggested that the mean fluorescence intensity (MFI) of intracellular ROS was significantly higher in neutrophil-like cells with NAP overexpression compared with the negative control (355.70 ± 20.10 vs 103.22 ± 4.71) (P< 0.001). In addition, Western blotting showed that the expressions of apoptosis-related protein Bax, active-caspase-3 and active-caspase-9 were all up-regulated in neutrophil-like cells with NAP overexpression compared with the negative control. CONCLUSION: NAP could promote the migration and ROS generation of neutrophil-like cells and accelerate the cell apoptosis.

First Report of Colletotrichum fructicola Causing Bitter Rot of Pear (Pyrus bretschneideri) in China.

Pyrus bretschneideri cv. Dangshansuli is the most important commercial Asiatic pear cultivar worldwide. In recent years, a fruit rot disease of unknown etiology have caused considerable fresh market losses in the 'Dangshansuli' production operations in Dangshan county, Anhui Province, China. Fresh market losses typically range from 60 to 90% and in 2008 were estimated at US$150 million. Symptomatic mature 'Dangshansuli' pears were collected from an orchard in Dangshan County in February 2008. A thin section (about 1 mm3) of symptomatic tissue was sterilized in a bleach and placed on potato dextrose agar (PDA) medium for isolation. From all fruit, a single fungus was recovered displaying gray-white dense aerial mycelium. Identical fungi were isolated from six additional symptomatic 'Dangshansuli' pears collected from other orchards in the county. Pathogenicity tests using one isolate (DS-0) were conducted in triplicate by placing 4 mm diameter discs from 7-day-old PDA plates onto the mature 'Dangshansuli' pear fruit that were incubated in an incubator at 25°C with a 12-h photoperiod for 30 days. An equal number of noncolonized PDA inoculations were included as a control. Isolate DS-0 caused symptoms similar to those in the field within 7 days and complete collapse of cortical tissues within 30 days. No symptoms were observed on control fruit. Round brownish lesions with a diameter of about 3 cm on inoculated fruit was populated by sunken, rotiform acervuli on which numerous, colorless, oblong single cell shape conidia with width/length of 6 × 20 µm were produced. A comparison of morphology and sequence analysis of the ribosomal internal transcribed spacer (ITS) regions in pre- and post-inoculation cultures from inoculated fruit confirmed the presence DS-0. To further characterize DS-0, aliquots of extracted genomic DNA from the fungus were subjected to PCR amplification and sequencing of seven gene regions from the ITS, actin (ACT), ß-tubulin 2 (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), manganese-superoxide dismutase (SOD2), chitin synthase (CHS-1), and calmodulin (CAL), using the primers listed by Weir et al (4), except for the primer pair of ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') for ITS amplification, and SODglo2-R (5'-TAGTACGCGTGCTCGGACAT-3') and SODglo2-R (5'-TAGTACGCGTGCTCGGACAT-3') for TBU2 amplification. Two or three clones of PCR products of each gene were sequenced and compared (GenBank Accession Nos. KC410780 to KC410786) to published data at http://www.cbs.knaw.nl/colletotrichum . The result indicated that DS-0 shared the highest similarity of 99.91% with Colletotrichum fructicola, corroborating numerous reports of Colletotrichum spp. causing bitter rot of pear on P. pyrifolia (1,2,3,4). C. fructicola was only recently reported as causing bitter rot of P. pyrifolia (4) and to our knowledge, this is the first report of C. fructicola causing bitter rot of P. bretschneideri, which will help producers select the best management practices for this devastating disease. References: (1) P. F. Cannon et al. Stud. Mycol. 73:181, 2012. (2) N. Tashiro et al. J. Gen. Plant Pathol. 78:221, 2012. (3) G. K. Wan et al. Mycobiology 35:238, 2007. (4) B. S. Weir et al. Stud. Mycol. 73:115, 2012.

Can surrounding greenery reduce noise annoyance at home?

Annoyance has been identified as the most important psychological impact arisen from noise. Besides socioeconomic status, residing neighborhood characteristics such as greenery has been shown to be able to reduce noise annoyance. To study the effects of these potential annoyance modifiers, nine hundred and ninety-two responses were collected through face-to-face interviews via questionnaire surveys. Among them, six-hundred and eighty-eight responses were collected together with adequate dwelling information which enabled a more accurate prediction of home noise levels. All these data were analyzed using an ordered logit model. Results indicate that greenery perception exerts considerable influence on noise annoyance rated at home. Wetland parks and garden parks are shown to be able to reduce noise annoyance to a greater degree than grassy hills. And the effects of the perceived amount of greenery on noise annoyance reduction at home differ according to the setting of greenery to which participant perceived from individual home.

Dynamic changes of gene expression profiles during postnatal development of the heart in mice.

OBJECTIVE: To study postnatal cardiac differentiation in the mouse. HYPOTHESIS: There might be mechanisms or factors in cardiac differentiation that could be identified by systematic gene expression analysis during postnatal cardiac development. METHODS: Expression of 6144 genes was examined in mouse heart, from the newborn period (day 0), through day 7 and day 14 day, to adulthood, using the cDNA microarray approach. Northern blotting and immunohistochemical techniques were used to confirm the microarray results. RESULTS: Various cardiac development related genes involving the cell cycle (cyclin B1, proliferating cell nuclear antigen (PCNA), and Ki67), growth factors (IGF-II, pleiotrophin (PTN), and midkine (MK)), and transcriptional regulation, cytoskeleton, and detoxification enzymes were identified by microarray analysis. Some of these genes were also confirmed by Northern blotting and immunohistochemistry of their RNA and protein content. In vivo treatment with PTN (20 ng/g) increased bromodeoxyuridine incorporation (by 2.24-fold) and PCNA expression (by 1.71-fold) during day 7 to day 14, indicating that PTN induces cell proliferation in mouse heart. CONCLUSIONS: Global gene expression analysis in the whole heart may be useful in understanding the orchestrated process of postnatal development or terminal differentiation in the cardiac environment. These data are likely to be helpful in studying developmental anomalies of the heart in neonates.

The voltammetric behavior of bone marrow of leukaemia and its clinical application.

The electrochemical voltammetric behavior of bone marrow of leukaemia has been investigated by a self-devised cytosensing system. The two anodic peaks of erythrocytes (red blood cells, RBC) in bone marrow of leukaemia appeared at 0.73 +/- 0.03 and 0.83 +/- 0.02V vs. SCE, respectively, on the first scan. The anodic peak of leukocytes (white blood cells, WBC) appeared at 0.32 +/- 0.03V vs. SCE. The anodic peak of RBC at 0.83V disappeared when the patients were cured. The experimental results show that the voltammetric behavior of erythrocytes is in constant contact with the initial stage of leukaemia. The cyclic voltammetric behavior of 40 cases of leukaemia including acute myeloid leukaemia (AML) and chronic myeloid leukaemia (CML) and 10 cases of healthy volunteer peripheral blood was studied. The cyclic voltammetric behavior of erythrocytes may provide a simple and specific marker for diagnosis of leukaemia.

Variable membrane glycoproteins in different growth cone populations.

The question of whether growth cones generated by different neurons contain distinctive membrane glycoproteins was examined. Growth cone particles (GCPs) were isolated from specific regions of fetal or early postnatal brain, and their membrane proteins were analyzed by 2D gel electrophoresis and Western blotting, using WGA as a probe. These blots were compared to those generated by synaptosomes from adult brain. The patterns reveal a number of WGA-binding glycoproteins that are uniformly present in these subcellular fractions and others that are found in GCPs from selected brain regions only. The results indicate, therefore, substantial pattern diversity for the different, restricted growth cone populations. Some of the WGA-binding glycoproteins seen in GCPs disappear with increasing age and are absent from synaptosomes, while others seem to become more prominent. One of the glycoprotein complexes present in all GCP and synaptosome fractions analyzed is gp93. It has an apparent molecular weight of 90-97 kDa and exhibits unusually high heterogeneity in GCPs from whole fetal brain. The gp93 complex covers a pI range from about 4.9 to about 6.4 and consists of at least 12 different species, probably isoelectric variants. In GCPs from different brain regions, the sets of gp93 species observed are different and characteristic. Neuraminidase digestion shifts the gp93 pattern to a more neutral pI but simplifies it only partially, indicating that variable sialic acid content explains the molecular diversity to some extent. Thus, gp93 is a glycoprotein complex whose members are expressed and/or posttranslationally processed differentially in different growth cone populations. Such a glycoprotein family may be involved in selective cell-cell recognition.

Identification of human N-ras as the common oncogene in NIH 3T3 cells transformed with DNAs from human primary hepatic cancer and hepatoma 7402 line.

DNA was extracted from NIH 3T3 cells transformed with DNAs from human primary hepatic cancer (PHC) and Hepatoma 7402 cell line. The transformant DNA was analyzed by Southern transfer and hybridization with 32P-labeled probes of various oncogenes. The EcoRI 7.2 and 9.0 kb bands characteristic of human N-ras gene were identified in transformed NIH 3T3 cells derived both from PHC and 7402 DNA. The BamHI 6.6 kb band characteristic of human c-Ha-ras I was present only in 7402 transformants, but not in PHC transformants. Using 35S-methionine incorporation, immunoprecipitation with anti-p21 monoclonal antibodies, SDS-PAGE and autoradiography, it was demonstrated that p21 synthesis was remarkably enhanced in 7402 cells as well as in transformed cells derived from both 7402 and PHC DNA. Taking the data together, it strongly implies that N-ras is one of the transforming genes for human liver cancer.