Objective To analyze the genomic sequences of Cryptococcus neoformans var grubii strains of two genotypes with different virulence and to screen out the virulence-associated genes. Methods A clinical strain (IFM56800) with the strongest virulence and an environmental strain (IFM56731) with the weakest virulence were screened out for whole genome sequencing analysis. The results of sequencing analy-sis were comprehensively analyzed by using the method of comparative genomics. Genetic variations were ex-tensively screened by using the strategies of non-synonymous single nucleotide polymorphisms ( nsSNPs), nonsense SNPs and the insertions or deletions ( InDels) causing frameshift mutations. The filtered genes were sequenced in 20 experimental strains. The whole RNAs were extracted and then the full-length cDNAs were sequenced by using the rapid amplification of 5′ and 3′ cDNA ends (RACE) method. Results By whole genome sequencing, valid data with high coverage (127 times and 111 times) was obtained in both the environmental strain IFM56731 and the clinical strain IFM56800. The data of InDels and SNPs were statisti-cally analyzed, respectively. Six genes were chosen for further analysis based on the strategies of nonsense SNPs and the InDels causing frameshift mutations. The six genes were amplified and sequenced in all of the experimental strains, three of which were further analyzed with cDNA sequencing. Ultimately, the location and structure of CNAG_01032 gene were determined. The predicted nonsense mutation locus was verified to present in the actual mRNA. Conclusion The strategies of nonsense SNPs and the InDels causing frame-shift mutations showed high-efficiency in screening potential virulence-associated genes. The CNAG_01032 gene was screened out as a novel virulence-associated gene.

Objective:To observe the effect of Lingshao-Zaoren Decoction on urodynamics and the expression of Piezo1 if overactive bladder (OAB) rats. Methods:Thirty SPF grade female SD rats were randomly divided into blank group, model group, Tolterodine control group, low-dose and high-dose Lingshao-Zaoren Decoction groups, with 6 rats in each group. The OAB rats were modeled by intraperitoneal injection of Cyclophosphamide. After the successful modeling, Tolterodine control group was given 0.36 mg/kg Tolterodine tartrate, the low-dose and high-dose Lingshao-Zaoren Decoction groups were given 1.59 and 3.18 g/kg Lingshao-Zaoren Mianjian granules by gavage, the blank group and model group were given the same amount of distilled water, once a day for 14 days. After 14 days, the urodynamics of rats in each group were detected. The bladder volume and maximum bladder pressure were observed respectively. The pathological changes of bladder tissue were observed by HE staining. The expression of Piezo1 protein in bladder tissue was detected by immunohistochemistry and Western blot. The expression of Piezo1 mRNA in bladder tissue was detected by qPCR. Results:Compared with the blank group, the body weight, bladder volume and maximum bladder pressure of the model group were significantly reduced ( P<0.01). HE staining result showed that the model group had hyperplasia of urinary tract epithelium, degeneration, necrosis and abscission of epithelial cells, infiltration of a large number of inflammatory cells in stroma, vascular proliferation, thickening of vascular wall, hyperplasia of mucosal smooth muscle, disorder of arrangement, and significant up regulation of Piezo1 protein expression ( P<0.01). Compared with the model group, the weight [(244.83 ± 6.05) g, (233.33 ± 11.76) g vs. (219.00 ± 9.70) g] of rats in the Tolterodine control group and high-dose group of Lingshao-Zaoren Decoction significantly increased ( P<0.01), and the bladder volume [(0.93 ± 0.31) ml, (1.17 ± 0.17) ml, (1.21 ± 0.23) ml vs. (0.50 ± 0.16) ml] and maximum bladder pressure [(42.00 ± 3.03) cmH 2O, (45.83 ± 7.19) cmH 2O, (46.83 ± 8.23) cmH 2O vs. (30.50 ± 5.47) cmH 2O] of rats in the Tolterodine control group, low-dose and high-dose Lingshao-Zaoren Decoction groups were significantly increased ( P<0.01); the bladder epithelial hyperplasia and degeneration degree, interstitial inflammatory cell infiltration degree and vascular hyperplasia degree of rats in the Tolterodine control group, low-dose and high-dose Lingshao-Zaoren Decoction groups significantly increased. The expression of Piezo1 mRNA (1.50 ± 0.04, 2.05 ± 0.08, 1.44 ± 0.10 vs. 2.56 ± 0.11) and protein in the Tolterodine control group, low-dose and high-dose Lingshao-Zaoren Decoction groups were significantly decreased ( P<0.01). Conclusion:Lingshao-Zaoren Decoction can increase the bladder volume and maximum bladder pressure of urinary incontinence caused by detrusor overactivity in rats with overactive bladder, which may be related to reduction of Piezo1 expression.

Objective To explore the effect of adipose-derived stem cells (ADSCs) on inflammatory factors in rats with lipopolysaccharide (LPS)-induced acute lung injury (ALI) and the possible mechanism of anti-inflammatory. Methods Seventy male Sprague-Dawley (SD) rats were randomly divided into normal control group (n = 10), LPS model group (n = 30), and ADSCs intervention group (n = 30) by random number table. ALI model was reproduced by intraperitoneal injection of 8 mg/kg LPS, and the rats in ADSCs intervention group received tail vein injection of 300 μL ADSCs 30 minutes after the model reproduction, the samples of normal control group were harvested immediately without any intervention, and the specimens in remained two groups were taken at 6, 24, 72 hours respectively. Arterial partial pressure of oxygen (PaO2) and lactate level in femoral artery were determined. Enzyme-linked immunosorbent assay (ELISA) was used to detect the serum myeloperoxidase (MPO) and interleukin-10 (IL-10) in the blood of left ventricle. Lung wet/dry weight (W/D) ratio was detected by thoracotomy, and the pathological changes of lung tissue were observed under an optical microscope. Western Blot was used to detect the protein expression of nuclear factor-κB (NF-κB) in lung tissue of rats. Results Compared with the normal control group, the damage degree of lung tissue of LPS model group was significantly heavier from 6 hours, and lung W/D ratio, blood lactate, MPO, IL-10 and expression level of NF-κB in lung tissue were significantly increased respectively, while PaO2 was decreased significantly. Compared with LPS model group, the damage degree of lung tissue of ADSCs intervention group was significantly reduced from 6 hours, and lung W/D ratio, blood lactate, MPO, and NF-κB expression in lung tissue were significantly decreased, while PaO2 was increased significantly, and it became normal at 72 hours [lung W/D ratio: 5.33±0.29 vs. 5.77±0.42 at 6 hours, 5.14±0.46 vs. 5.43±0.38 at 72 hours; blood lactate (mmol/L): 3.6±1.0 vs. 5.7±1.1 at 6 hours, 3.1±1.0 vs. 3.8±1.2 at 72 hours; blood MPO (μg/L): 1.50±0.90 vs. 2.70±1.85 at 6 hours, 0.46±0.30 vs. 0.71±0.22 at 72 hours; NF-κB (gray value): 0.40±0.11 vs. 0.50±0.09 at 6 hours, 0.24±0.03 vs. 0.33±0.06; PaO2 (mmHg, 1 mmHg = 0.133 kPa): 78.0±4.1 vs. 74.5±3.2 at 6 hours, 89.3±9.4 vs. 81.9±3.4 at 72 hours; all P < 0.05]. The IL-10 level was significantly higher than that of LPS model group only at 24 hours (ng/L: 27.75±15.49 vs. 17.52±6.56, P < 0.05). Conclusion ADSCs can effectively relieve the inflammatory response of ALI induced by LPS, probably by inhibiting the expressions of NF-κB and blocking the release of inflammatory cytokines.

ObjectiveTo investigate the difference in protein expression between hepatocellular carcinoma (HCC) patients with recurrence and those with good prognosis, the differential expression and regulatory mechanism of miR-152-3p target proteins, and the role of miR-152-3p in the recurrence of HCC. MethodsTMT-labeled proteomic sequencing and RT-PCR were used to measure the expression of proteins and the expression of miR-152-3p in the HCC tissue of six patients with recurrence at 2 years after HCC resection and six patients with good prognosis at 5 years. Six databases were used to analyze the target genes of miR-152-3p, and Gene Ontology, DAVID, and REACTOME databases were used to perform target gene screening, enrichment annotation, and signal transduction pathway enrichment analysis. Gene mutation frequency and survival curve analysis were performed for the target genes of miR-152-3p to verify the role of miR-152-3p target genes in patients with HCC recurrence. The independent samples t-test was used for comparison of continuous data between two groups, and a Kaplan-Meier analysis was performed to investigate the survival rates of liver-related genes. ResultsCompared with the patients with HCC recurrence, the patients with good prognosis after HCC resection had a significantly higher transcriptional expression level of miR-152-3p in HCC tissue (P＜0.05). The results of protein sequencing showed that there were 365 differentially expressed proteins in HCC tissue between the patients with good prognosis and the patients with recurrence, and the analysis of HCC recurrence databases showed that 17 proteins were regulated by miR-152-3p. Further analysis of the signaling pathways showed that the function of the 17 target genes regulated by miR-152-3p was enriched in the translation and regulation of mitochondria and ribosome, and multiple enrichment revealed that six target genes were closely associated with mitochondrial respiratory chain complex, i.e., AKAP1, FOXRED1, MRPL28, MRPL50, SHC1, and STAU1. Gene mutation frequency and survival curve analysis showed that the loss or weakening of the function of mitochondrial respiratory chain-related target proteins seriously affected the prognosis and survival rate of patients. ConclusionThere is a significant difference in the expression of miR-152-3p in HCC tissue between patients with good prognosis and those with recurrence after HCC resection, and miR-152-3p may lead to the recurrence of HCC by regulating the target genes AKAP1, FOXRED1, MRPL28, MRPL50, SHC1, and STAU1, acting on the mitochondrial respiratory chain, and affecting the oxidative respiratory function of cells.

Objective To explore the effect of 5-ethynyl-2'-deoxyuridine (EdU) -labeled adipose-derived stem cells (ADSCs) on lung colonization, TNF-α and IL-4 in rats induced by lipopolysaccharide (LPS) with acute lung injury. Methods Thirty male Sprague-Dawley (SD) rats were randomly divided into the normal control group (n=10), LPS model group (n=10), and LPS+ADSCs intervention group (n=10). The ALI model rats were intraperitoneally injected with 8 mg/kg LPS, rats in the normal control group were intraperitoneally injected with 4 mL/kg physiological saline, and rats in the LPS+ADSCs group were intravenously injected with 300 μL ADSCs by tail vein after 30 minutes for the ALI model establishment, and rats in the normal control group and LPS group were intravenously injected with 300μL physiological saline by tail vein. The time of death in rats was observed, lung tissue and blood from left ventricular were collected, and the serum tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-4) were detected by Enzyme-linked immunosorbent assay (ELISA). Lung wet/dry weight (W/D) ratio was detected by thoracotomy, the pathological changes of lung tissue were observed under optical microscope, and the colonization of ADSCs in the lungs were observed under immunofluorescence microscopy. LSD-t method was used to compare between every two groups. Results There was no significant difference in mortality between the LPS group and LPS + ADSCs group (50％ vs. 70％, P> 0.05); EdU-labeled ADSCs were extensively colonized in the lungs by tail vein injection after 24 h; Compared with the normal control group, the lung injury of the LPS group was heavier, the ratio of lung W/D and TNF-α were significantly increased (all P< 0.01), and IL-4 level was significantly decreased (P< 0.01). Compared with the LPS model group, the degree of lung injury in the LPS + ADSCs group was significantly reduced, lung W/D ratio (5.57±0.27 vs. 5.98±0.28) and TNF-α level of blood [(41.51±4.14)ng/L vs. (45.52±3.74)ng/L] were significantly reduced (all P< 0.05), whereas the IL-4 levels were significantly increased [(7.01±1.11)pg/mL vs. (3.27±0.54)pg/mL, P< 0.05]. Conclusions EdU-labeled ADSCs could be colonized in the lungs of LPS-induced ALI rats, reduce the inflammatory response from TNF-α and improve the anti-inflammatory response from IL-4.

Objective: To evaluate the efficacy of Fufang-Xuanju capsule combined with levofloxacin mesylate tablets in the treatment of chronic epididymitis. Methods: A total of 76 patients in the Urology Department of Xiyuan Hospital of China Academy of Chinese Medical Sciences who met the inclusion criteria from December 2016 to February 2019, were divided into treatment group (43 cases) and control group (33 cases). The control group was given levofloxacin mesylate tablets orally. The treatment group was given Fufang-Xuanju capsule based on the control group. Both groups were treated for 4 weeks. Chronic epididymitis symptom index (CESI) was used to evaluate the clinical efficacy by pain score and quality of life score. Results: The total effective rate was 83.7% (36/43) in the treatment group and 63.6% (26/33) in the control group. There was statistically significant difference between the two groups (χ²=4.020, P=0.045). Compared with baseline, the pain scores, quality of life scores and total scores of both groups at 2, 4 weeks after treatment were significantly lower (P<0.01). Two weeks after the treatment, the pain scores, quality of life scores and total scores in the treatment group were significantly lower than those of the control group (F value were 16.132, 9.134 and 23.681, respectively, all Ps<0.01). And 4 weeks after the treatment, the pain scores, quality of life scores and total scores of the treatment group were significantly lower than that of the control group (F value were 28.741, 74.049, 72.483, respectively, all Ps<0.01). Conclusions Fufang-Xuanju capsule combined with levofloxacin mesylate tablets can alleviate the pain of patients with chronic epididymitis, improve the quality of life and improve the clinical efficacy.