Clinical value of contrast-enhanced ultrasound versus conventional ultrasound in biopsy of focal liver lesions.

BACKGROUND: Focal liver lesions (FLLs) are a common form of liver disease, and identifying accurate pathological types is required to guide treatment and evaluate prognosis. PURPOSE: To compare and analyze the application effect of contrast-enhanced ultrasound (CEUS) and conventional ultrasound (US) in the clinical diagnosis of focal liver lesions. MATERIAL AND METHODS: A retrospective analysis was performed on 682 patients with space-occupying liver lesions admitted to our hospital between December 2015 and August 2021. Of these, 280 underwent CEUS-guided biopsies and 402 underwent conventional US biopsies, with the results of each biopsy subsequently compared between the two groups. The success rate and accuracy of the biopsies and their relationship with different pathological features were also analyzed. RESULTS: The success rate, sensitivity, diagnostic accuracy, positive predictive value, and negative predictive value of the CEUS group were significantly higher than those of the US group (P < 0.05). Lesion size accuracy in the CEUS group was significantly higher than that in the US group (89.29% vs. 40.55%; P < 0.05). Lesion type accuracy in the CEUS group was significantly higher than that in the US group (86.49% vs. 43.59%), and the difference between the two groups was statistically significant (P < 0.05). The logistic regression analysis indicated that malignant lesions, lesions ≥5 cm, and lesions ≤1 cm were independent factors affecting the success rate of the puncture procedure (P < 0.05). CONCLUSION: The sensitivity, specificity, and diagnostic accuracy of lesion size and type in the CEUS group were higher than those in the US group.

Dietary Methionine Restriction Improves Gut Health and Alters the Plasma Metabolomic Profile in Rats by Modulating the Composition of the Gut Microbiota.

Dietary methionine restriction (MetR) offers an integrated set of beneficial health effects, including delaying aging, extending health span, preventing fat accumulation, and reducing oxidative stress. This study aimed to investigate whether MetR exerts entero-protective effects by modulating intestinal flora, and the effect of MetR on plasma metabolites in rats. Rats were fed diets containing 0.86% methionine (CON group) and 0.17% methionine (MetR group) for 6 weeks. Several indicators of inflammation, gut microbiota, plasma metabolites, and intestinal barrier function were measured. 16S rRNA gene sequencing was used to analyze the cecal microbiota. The MetR diet reduced the plasma and colonic inflammatory factor levels. The MetR diet significantly improved intestinal barrier function by increasing the mRNA expression of tight junction proteins, such as zonula occludens (ZO)-1, claudin-3, and claudin-5. In addition, MetR significantly increased the levels of short-chain fatty acids (SCFAs) by increasing the abundance of SCFAs-producing Erysipclotxichaceae and Clostridium_sensu_stricto_1 and decreasing the abundance of pro-inflammatory bacteria Proteobacteria and Escherichia-Shigella. Furthermore, MetR reduced the plasma levels of taurochenodeoxycholate-7-sulfate, taurocholic acid, and tauro-ursodeoxycholic acid. Correlation analysis identified that colonic acetate, total colonic SCFAs, 8-acetylegelolide, collettiside I, 6-methyladenine, and cholic acid glucuronide showed a significant positive correlation with Clostridium_sensu_stricto_1 abundance but a significant negative correlation with Escherichia-Shigella and Enterococcus abundance. MetR improved gut health and altered the plasma metabolic profile by regulating the gut microbiota in rats.

Clinical implications of micro lymph node metastasis for patients with gastric cancer.

BACKGROUND: Lymph node size is considered as a criterion for possible lymph node metastasis in imageology. Micro lymph nodes are easily overlooked by surgeons and pathologists. This study investigated the influencing factors and prognosis of micro lymph node metastasis in gastric cancer. METHODS: 191 eligible gastric cancer patients who underwent D2 lymphadenectomy from June 2016 to June 2017 in the Third Surgery Department at the Fourth Hospital of Hebei Medical University were retrospectively analyzed. Specimens were resected en bloc and the postoperative retrieval of micro lymph nodes was carried out by the operating surgeon for each lymph node station. Micro lymph nodes were submitted for pathological examination separately. According to the results of pathological results, patients were divided into the "micro-LNM (micro lymph node metastasis)" group (N = 85) and the "non micro-LNM" group (N = 106). RESULTS: The total number of lymph nodes retrieved was 10,954, of which 2998 (27.37%) were micro lymph nodes. A total of 85 (44.50%) gastric cancer patients had been proven to have micro lymph node metastasis. The mean number of micro lymph nodes retrieved was 15.7. The rate of micro lymph node metastasis was 8.1% (242/2998). Undifferentiated carcinoma (90.6% vs. 56.6%, P = 0.034) and more advanced Pathological N category (P < 0.001) were significantly related to micro lymph node metastasis. The patients with micro lymph node metastasis had a poor prognosis (HR for OS of 2.199, 95% CI = 1.335-3.622, P = 0.002). For the stage III patients, micro lymph node metastasis was associated with shorter 5-year OS (15.6% vs. 43.6%, P = 0.0004). CONCLUSIONS: Micro lymph node metastasis is an independent risk factor for poor prognosis in gastric cancer patients. Micro lymph node metastasis appears to be a supplement to N category in order to obtain more accurate pathological staging.

Tuning crystal orientation and chiral spin order in Mn3Ge by annealing process and ion implantation.

The non-collinear antiferromagnetic Weyl semimetal Mn3X (X = Ga, Ge, Sn) system has attracted a lot of attentions owing to its robust anomalous Hall effect (AHE), large spin Hall angle and small net magnetization at room temperature. The high spin-charge interconversion efficiency makes it a super candidate in topological antiferromagnetic spintronic devices, which could facilitate ultra-fast operation of high-density devices with low energy consumption. In this work, we have realized to obtain different chiral spin structures in Heusler alloy Mn3Ge thin films, which originate from different crystalline orientations. The high-quality (0002)- and (202¯0)-oriented single phase hexagonal Mn3Ge films are achieved by controllable growth, annealing process and ion implantation. The various magnetic properties and AHE behaviors are observed alongaandccrystal axes, equivalent to magnetic field in and out of the inverse triangular spin plane. The observation demonstrates the manipulation of crystal structure accompanied with chiral spin order in a non-collinear antiferromagnetic Mn3Ge film, which is induced by energy conversion and defect introduction. Thein situthermal treatment induces crystal phase rotation up to 90° and robust AHE modulation, which is significantly important and highly desirable for flexible spin memory device applications.

Exploring the Metal-Insulator Transition in (Ga,Mn)As by Molecular Absorption.

The metal-insulator transition (MIT) is normally assisted by certain external power input, such as temperature, pressure, strain, or doping. However, these may increase the disorder of the crystal or cause other effects, which makes device fabrication complicated and/or hinders large-scale application. Here, we adopt a new approach to obtain robust modulation of physical properties in magnetic semiconductor (Ga,Mn)As by surface molecular modification. We have probed both sides of the MIT with n- and p-type molecular doping. Density functional theory calculations are carried out to determine the stable absorption configuration and charge transfer mechanism of electron acceptor and donor molecules on the semiconductor surface. Both experimental and theoretical results confirm a remarkable modulation in carrier concentrations without introducing impurities or defects. This work points out the possibility of effectively tuning physical properties of solid-state materials by functional molecules, which is clean, flexible, nondestructive, and easily achieved.

Analysis of transcriptome differences between subcutaneous and intramuscular adipose tissue of Ningxiang pigs.

To compare and analyze the molecular mechanisms of adipose deposition in subcutaneous fat (SAF)and intramuscular fat (IMF) tissues in Ningxiang pigs, differential gene expression profiles in SAF and IMF tissues of Ningxiang pigs were identified and analysed using RNA-seq technology. Six healthy 250-day-old male Ningxiang pigs with similar body weights (approximately 85 kg) of intraspecific individuals were selected as experimental material and samples of SAF and IMF tissues were collected. Differential genes associated with fat deposition and lipid metabolism were obtained by sequencing two adipose tissue transcriptomes and performing GO (Gene Ontology) functional annotation and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis. To verify the reliability of the sequencing results, six differential genes were randomly selected to validate using qRT-PCR. The results showed that we identified 2406 DEGs, with 1422 up-regulated and 984 down-regulated genes in two tissues. GO functional annotation analysis revealed that the differentially expressed genes were mainly involved in lipid metabolism related pathways, such as steroid biosynthesis, unsaturated fatty acid biosynthesis, glycerophospholipid metabolism and autophagy pathway. KEGG pathway enrichment showed that the differentially expressed genes were mainly enriched in the biological processes related to lipid binding, fatty acid metabolism, glycol ester metabolism, lipid biosynthesis and other biological processes related to lipid metabolism. Genes related to lipid metabolism, such as TCAP, NR4A1, ACACA, LPL, ELOVL6, DGAT1, PRKAA1, ATG101, TP53INP2, FDFT1, ACOX1 and SCD were identified by bioinformatic analyses and verified by qRT-PCR. Our results indicated that these genes may play important roles in the regulation of fat deposition and metabolism in the SAF and IMF tissue, providing the further mechanistic investigation of fat deposition in Ningxiang pigs.

IKAROS and CK2 regulate expression of BCL-XL and chemosensitivity in high-risk B-cell acute lymphoblastic leukemia.

High-risk B-cell acute lymphoblastic leukemia (B-ALL) is an aggressive disease, often characterized by resistance to chemotherapy. A frequent feature of high-risk B-ALL is loss of function of the IKAROS (encoded by the IKZF1 gene) tumor suppressor. Here, we report that IKAROS regulates expression of the BCL2L1 gene (encodes the BCL-XL protein) in human B-ALL. Gain-of-function and loss-of-function experiments demonstrate that IKAROS binds to the BCL2L1 promoter, recruits histone deacetylase HDAC1, and represses BCL2L1 expression via chromatin remodeling. In leukemia, IKAROS' function is impaired by oncogenic casein kinase II (CK2), which is overexpressed in B-ALL. Phosphorylation by CK2 reduces IKAROS binding and recruitment of HDAC1 to the BCL2L1 promoter. This results in a loss of IKAROS-mediated repression of BCL2L1 and increased expression of BCL-XL. Increased expression of BCL-XL and/or CK2, as well as reduced IKAROS expression, are associated with resistance to doxorubicin treatment. Molecular and pharmacological inhibition of CK2 with a specific inhibitor CX-4945, increases binding of IKAROS to the BCL2L1 promoter and enhances IKAROS-mediated repression of BCL2L1 in B-ALL. Treatment with CX-4945 increases sensitivity to doxorubicin in B-ALL, and reverses resistance to doxorubicin in multidrug-resistant B-ALL. Combination treatment with CX-4945 and doxorubicin show synergistic therapeutic effects in vitro and in preclinical models of high-risk B-ALL. Results reveal a novel signaling network that regulates chemoresistance in leukemia. These data lay the groundwork for clinical testing of a rationally designed, targeted therapy that combines the CK2 inhibitor, CX-4945, with doxorubicin for the treatment of hematopoietic malignancies.

LncRNA MEG8 plays an oncogenic role in hepatocellular carcinoma progression through miR-367-3p/14-3-3ζ/TGFßR1 axis.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and it carries a poor prognosis. Clarifying the pathologic mechanisms of this disease will be beneficial for the diagnosis and treatment of HCC. LncRNA MEG8 is involved in several tumors but its role in HCC progression remains unknown. This study was designed to explore the role and regulatory mechanisms of MEG8 in HCC progression. MTT, EdU, wound-healing, and transwell assays were employed to analyze the proliferation, migration, and invasion of HCC cells. A luciferase assay was utilized to confirm the predicted binding site. RNA immunoprecipitation and co-immunoprecipitation were employed to verify the binding between MEG8 and miR-367-3p as well as 14-3-3ζ and TGFßR1. Real-time PCR and western blot were employed to detect the expression of interesting genes. Results revealed that MEG8 was increased in HCC tissues and cells, and was correlated with the poor prognosis of HCC patients. Inhibiting MEG8 significantly repressed the HCC cells' ability to proliferate, migrate, and invade. Moreover, MEG8 sponged miR-367-3p to upregulate 14-3-3ζ, the binding of which suppressed TGFßR1 degradation, thereby enhancing TGFß signaling. In conclusion, this work exposed a novel role and regulatory mechanism of MEG8 in HCC and provided new insight into the treatment of HCC.

A novel G6PD deleterious variant identified in three families with severe glucose-6-phosphate dehydrogenase deficiency.

BACKGROUND: Glucose-6-phosphate dehydrogenase deficiency (D-G6PD) is an X-linked recessive disorder resulted from deleterious variants in the housekeeping gene Glucose-6-phosphate 1-dehydrogenase (G6PD), causing impaired response to oxidizing agents. Screening for new variations of the gene helps with early diagnosis of D-G6PD resulting in a reduction of disease related complications and ultimately increased life expectancy of the patients. METHODS: One thousand five hundred sixty-five infants with pathological jaundice were screened for G6PD variants by Sanger sequencing all of the 13 exons, and the junctions of exons and introns of the G6PD gene. RESULTS: We detected G6PD variants in 439 (28.1%) of the 1565 infants with pathological jaundice. In total, 9 types of G6PD variants were identified in our cohort; and a novel G6PD missense variant c.1118 T > C, p.Phe373Ser in exon 9 of the G6PD gene was detected in three families. Infants with this novel variant showed decreased activity of G6PD, severe anemia, and pathological jaundice, consistent with Class I G6PD deleterious variants. Analysis of the resulting protein's structure revealed this novel variant affects G6PD protein stability, which could be responsible for the pathogenesis of D-G6PD in these patients. CONCLUSIONS: High rates of G6PD variants were detected in infants with pathological jaundice, and a novel Class I G6PD deleterious variants was identified in our cohort. Our data reveal that variant analysis is helpful for the diagnosis of D-G6PD in patients, and also for the expansion of the spectrum of known G6PD variants used for carrier detection and prenatal diagnosis.

Dynamic changes in circulating levels of metabolites in the portal-drained viscera of finishing pigs receiving acute administration of l-arginine.

In this study, we examined the effects of acute intravenous administration of l-arginine on circulating levels of metabolites in the portal-drained viscera (PDV) of 12 barrows surgically fitted with chronic catheters in the portal vein. At day 14 post-surgery, the pigs were fasted for 12 hr and then randomly allocated to one of three groups to receive administration of normal saline, l-alanine [103 mg/kg body weight (BW), isonitrogenous control] or l-arginine-HCl (61 mg/kg BW), via the portal vein. Blood samples were obtained from the carotid artery before and at 30-min intervals for 5 hr after the administration of saline or amino acid in order to determine metabolic profiles. The results showed that, compared with the saline treatment, arginine infusion increased plasma concentrations of insulin-like growth factor-I, arginine and cystine in the portal vein plasma, whereas plasma concentrations of threonine, serine, leucine and methionine were reduced. These findings indicate that increasing arginine concentrations in the portal vein alters the metabolic profile in swine, an established animal model for studying human nutrition and metabolism.

The Concentration of Iodine in Perigastric Adipose Tissue: A Novel Index for the Assessment of Serosal Invasion in Patients with Gastric Cancer after Neoadjuvant Chemotherapy.

OBJECTIVE: This study aims to explore the association between iodine concentration (IC) in perigastric adipose tissue (PAT), quantified by dual-energy computed tomography (DECT) and serosal invasion (SI) in patients with gastric cancer post-neoadjuvant chemotherapy (NAC). METHODS: Forty-three patients with T4-staged gastric cancer were enrolled. IC and standardized IC in PAT (ICPAT and SICPAT) were quantified by DECT pre and post NAC. A postoperative pathologic examination was performed to stage gastric cancer. RESULTS: After NAC, a total of 43 participants were assigned to group A with 13 patients and group B with 30 patients according to the results of the postoperative pathologic examination. The accuracy of conventional CT in identifying SI was 74.42%. Differences of variations between pre- and post- NAC ICPAT, SICPAT, ∆ICPAT, and ∆SICPAT were observed respectively (p < 0.05). Intragroup ICPAT and SICPAT also changed significantly after NAC (p < 0.05). The area under the ROC curve was 0.929, with the threshold of ∆SICPAT reaching 0.095. The sensitivity, specificity, and accuracy of SICPAT in identifying post-NAC SI were 92.30, 86.70, and 88.37% respectively. Moreover, the 2 measurements in the same patient maintain a high level of consistency. CONCLUSION: These results showed that SICPAT is a reliable index for identifying post-NAC SI.

Transcriptional Regulation of JARID1B/KDM5B Histone Demethylase by Ikaros, Histone Deacetylase 1 (HDAC1), and Casein Kinase 2 (CK2) in B-cell Acute Lymphoblastic Leukemia.

Impaired function of the Ikaros (IKZF1) protein is associated with the development of high-risk B-cell precursor acute lymphoblastic leukemia (B-ALL). The mechanisms of Ikaros tumor suppressor activity in leukemia are unknown. Ikaros binds to the upstream regulatory elements of its target genes and regulates their transcription via chromatin remodeling. Here, we report that Ikaros represses transcription of the histone H3K4 demethylase, JARID1B (KDM5B). Transcriptional repression of JARID1B is associated with increased global levels of H3K4 trimethylation. Ikaros-mediated repression of JARID1B is dependent on the activity of the histone deacetylase, HDAC1, which binds to the upstream regulatory element of JARID1B in complex with Ikaros. In leukemia, JARID1B is overexpressed, and its inhibition results in cellular growth arrest. Ikaros-mediated repression of JARID1B in leukemia is impaired by pro-oncogenic casein kinase 2 (CK2). Inhibition of CK2 results in increased binding of the Ikaros-HDAC1 complex to the promoter of JARID1B, with increased formation of trimethylated histone H3 lysine 27 and decreased histone H3 Lys-9 acetylation. In cases of high-risk B-ALL that carry deletion of one Ikaros (IKZF1) allele, targeted inhibition of CK2 restores Ikaros binding to the JARID1B promoter and repression of JARID1B. In summary, the presented data suggest a mechanism through which Ikaros and HDAC1 regulate the epigenetic signature in leukemia: via regulation of JARID1B transcription. The presented data identify JARID1B as a novel therapeutic target in B-ALL and provide a rationale for the use of CK2 inhibitors in the treatment of high-risk B-ALL.

Targeting casein kinase II restores Ikaros tumor suppressor activity and demonstrates therapeutic efficacy in high-risk leukemia.

Ikaros (IKZF1) is a tumor suppressor that binds DNA and regulates expression of its target genes. The mechanism of Ikaros activity as a tumor suppressor and the regulation of Ikaros function in leukemia are unknown. Here, we demonstrate that Ikaros controls cellular proliferation by repressing expression of genes that promote cell cycle progression and the phosphatidylinositol-3 kinase (PI3K) pathway. We show that Ikaros function is impaired by the pro-oncogenic casein kinase II (CK2), and that CK2 is overexpressed in leukemia. CK2 inhibition restores Ikaros function as transcriptional repressor of cell cycle and PI3K pathway genes, resulting in an antileukemia effect. In high-risk leukemia where one IKZF1 allele has been deleted, CK2 inhibition restores the transcriptional repressor function of the remaining wild-type IKZF1 allele. CK2 inhibition demonstrated a potent therapeutic effect in a panel of patient-derived primary high-risk B-cell acute lymphoblastic leukemia xenografts as indicated by prolonged survival and a reduction of leukemia burden. We demonstrate the efficacy of a novel therapeutic approach for high-risk leukemia: restoration of Ikaros tumor suppressor activity via inhibition of CK2. These results provide a rationale for the use of CK2 inhibitors in clinical trials for high-risk leukemia, including cases with deletion of one IKZF1 allele.

Upregulated Vav2 in gastric cancer tissues promotes tumor invasion and metastasis.

Several studies have proved that Vav2 gene is associated with the carcinogenesis of some tumors, but the relationship between Vav2 gene and gastric cancer remains unclear. Purpose of this study is to detect the expression of Vav2 protein in gastric cancer tissues and to evaluate the clinical value of Vav2. Furthermore, both effect of Vav2 gene on invasion and metastasis of gastric cancer cells and its mechanism are investigated in vitro. Results showed that positive rate of Vav2 protein was significantly higher in gastric cancer tissues than in adjacent tissues and notably higher in metastatic lymph nodes than in gastric cancer tissues. Results of western blot were consistent with immunohistochemistry. Expression of Vav2 protein in gastric cancer tissues was related to degree of tumor differentiation, lymph node metastasis, and clinical stages. Inhibition of endogenous Vav2 in BGC823 cells led to significantly decreased cell activity, migration, and invasion ability in vitro, and expression of Rac1, MMP-2, and MMP-9 decreased, whereas expression of TIMP-1 increased. We concluded that Vav2 might promote invasion and metastasis of gastric cancer by regulating some invasion and metastasis-related genes.

Inhibition of Vav3 gene can promote apoptosis of human gastric cancer cell line MGC803 by regulating ERK pathway.

Previous studies proved that Vav3 gene was overexpressed in cancers. However, the molecular mechanism of Vav3 in apoptosis still keeps unclear; therefore, the relationship between Vav3 gene and apoptosis of gastric cancer (GC) was explored in the present study. Vav3-siRNA was transfected into MGC803 cells, and then cell activity and apoptosis rate were tested with MTT and FCM; apoptosis-related genes and proteins in MAPK signaling pathway were also tested. Results showed that Vav3 was overexpressed in GC than in adjacent normal tissues (all P < 0.05), and expression of Vav3 was related to degree of histological differentiation, cancer invasion depth, and lymphatic metastasis (Χ (2) = 7.185, P = 0.007; Χ (2) = 18.654, P < 0.001; Χ (2) = 5.058, P = 0.025). Vav3 silencing inhibited activity of MGC803 cells, and apoptosis rate of cells was affected. Vav3-siRNA transfection led to changes of apoptosis-related genes such as Survivin, xIAP, Bcl-2, caspase-3, and Bax (all P < 0.01). After transfection, ratio of phosphorylation of ERK significantly reduced. We concluded that Vav3 inhibition can suppress cell activity and promote apoptosis by regulating the apoptosis-related genes through the ERK pathway.

Naringenin inhibits proliferation, migration, and invasion as well as induces apoptosis of gastric cancer SGC7901 cell line by downregulation of AKT pathway.

The preliminary anti-cancer activity of Naringenin (Nar) has been proven in several cancers. However, the therapeutic activity of Nar on gastric cancer SGC-7901 cell line is not yet well understood. The aim of the present study was to investigate the effect and mechanisms of Nar on proliferation, apoptosis, migration, and invasion of SGC-7901 cells. In this in vitro study, SGC-7901 cells were treated with Nar at serial concentrations. Our data showed that Nar efficiently inhibited SGC-7901 cell proliferation in a time- and concentration-dependent manner, as well as downregulated proliferating cell nuclear antigen (PCNA) levels in a concentration-dependent manner. Meanwhile, the cell migration and invasion also dramatically decreased after Nar incubation, and the expressions of MMP2 and MMP9 were significantly downregulated. In addition, a strong proapoptotic effect was observed in the SGC-7901 cells after Nar treatment. Apoptosis-related proteins Bax and cleaved caspase-3 were up-regulated, whereas Bcl-2 and Survivin were downregulated. After administration with Nar, we found that phosphorylation of AKT was inhibited, and this inhibitory action could be mildly enhanced by the combination treatment of Nar and AKT inhibitor LY294002. In conclusion, our study confirmed that Nar could inhibit SGC-7901cell proliferation, migration, and invasion as well as induces apoptosis, and Nar might provide a new potential therapeutic strategy for treating gastric cancer.

[Comparison of Six Flavonoid Components of Closely-Related Plants Agrimonia pilosa,Potentilla chinensis and Potentilla discolor].

Objective: To establish an UHPLC method for simultaneous determination of six flavonoid components of three closelyrelated plants Agrimonia pilosa,Potentilla chinensis and Potentilla discolor including rutin,hyperoside,cynaroside,quercetin,apigenin and kaempferol. Meanwhile three fresh and dry plants were evaluated to compare the contents of six flavonoid components. Methods: The samples were pretreated with ultrasonic extraction with 70% ethanol for 0. 5 h. The analysis was performed on an Acquity HSS T3( 100 mm × 3. 0 mm,1. 8 µm) column with the mobile phase consisting of acetonitrile and 0. 3% glacial acetic acid aqueous at a flow rate of0. 4 m L / min. The detection wavelength was 360 nm,and the column temperature was 35 ℃. Results: The contents of rutin and hyperoside were high generally,but the content of kaempferol was extremely low in three closely-related plants. The content of apigenin was0. 028 mg / g in Potentilla chinensis,but not detected in Agrimonia pilosa and Potentilla discolor. The content of cymaroside in Agrimonia pilosa was significantly higher than that in Potentilla chinensis and Potentilla discolor. The fresh plants of Potentilla chinensis and Potentilla discolor contained more flavonoids than oven drying plants. Conclusion: The similar trend of content change from fresh to dry plant has showed a chemotaxonomic relationship of Potentilla chinensis and Potentilla discolor. The established determination method is simple,rapid and efficient,and is applicable for analysis of the contents of flavonoids in three closely-related plants,which provides the scientific basis for rationalization of using these drugs in clinic.

[Construction of expression vector pLCK-CD69-IRES-EGFP and generation of CD69 transgenic mice].

OBJECTIVE: To construct the expression vector pLCK-CD69-IRES-EGFP that contains mouse cell surface activation protein CD69 and enhanced green fluorescent protein(EGFP),and to generate CD69 transgenic mice based on this vector. METHODS: First, RNA was extracted from mouse lung tissue and cDNA was synthesized via reverse transcription. PCR primer was designed through the PubMed searching, then mouse CD69 DNA fragment was amplified with PCR. Second, this DNA fragment was subcloned to the pInsulater-LCK-IRES-EGFP plasmid and constructed the transgenic vector after the verification of nucleotide sequence. Third, the expression vector was then transfected into 293 T cells and its expression in 293 T cells was observed under fluorescence microscope. Last, microinjection was performed to transfer the expression vector pLCK-CD69-IRES-EGFP into fertilized eggs, which were implanted into pseudo-pregnant recipient mice. After birth the tail samples of the pups were obtained for the purpose of genotyping to determine the transgenic founders. Fluorescence microscope and flow cytometer were used to measure the expression of CD69 on cells. RESULTS: The construction of the expression vector pLCK-CD69-IRES-EGFP was verified by enzyme digestion and DNA sequencing. The transfected 293 T cell showed expression of the protein under fluorescence microscope. Identification of PCR for the tail tissue of the pups confirmed the present of CD69 transgene and resting lymphocytes demonstrated the expression of CD69. CONCLUSION: The construction of expression vector pLCK-CD69-IRES-EGFP and generation of CD69 transgenic mice have been successfully processed, which lays a foundation of the solid pattern studies in inflammatory diseases.

The interaction of caveolin 3 protein with the potassium inward rectifier channel Kir2.1: physiology and pathology related to long qt syndrome 9 (LQT9).

Mutations in CAV3 cause LQT syndrome 9 (LQT9). A previously reported LQT9 patient had prominent U waves on ECG, a feature that has been correlated with Kir2.1 loss of function. Our objective was to determine whether caveolin 3 (Cav3) associates with Kir2.1 and whether LQT9-associated CAV3 mutations affect the biophysical properties of Kir2.1. Kir2.1 current (IK1) density was measured using the whole-cell voltage clamp technique. WT-Cav3 did not affect IK1. However, F97C-Cav3 and T78M-Cav3 decreased IK1 density significantly by ∼60%, and P104L-Cav3 decreased IK1 density significantly by ∼30% at -60 mV. Immunostained rat heart cryosections and HEK293 cells cotransfected with Kir2.1 and WT-Cav3 both demonstrated colocalization of Kir2.1 and WT-Cav3 by confocal imaging. Cav3 coimmunoprecipitated with Kir2.1 in human ventricular myocytes and in heterologous expression systems. Additionally, FRET efficiency was highly specific, with a molecular distance of 5.6 ± 0.4 nm, indicating close protein location. Colocalization experiments found that Cav3 and Kir2.1 accumulated in the Golgi compartment. On-cell Western blot analysis showed decreased Kir2.1 cell surface expression by 60% when expressed with F97C-Cav3 and by 20% when expressed with P104L-Cav3 compared with WT-Cav3. This is the first report of an association between Cav3 and Kir2.1. The Cav3 mutations F97C-Cav3, P104L-Cav3, and T78M-Cav3 decreased IK1 density significantly. This effect was related to a reduced cell surface expression of Kir2.1. Kir2.1 loss of function is additive to the increase described previously in late INa, prolonging repolarization and leading to arrhythmia generation in Cav3-mediated LQT9.

Regulatory mechanism of ZNF139 in multi-drug resistance of gastric cancer cells.

Our previous study found increased zinc finger protein 139 (ZNF139) expression in gastric cancer (GC) cells. Purpose of the study is to further clarify the role and mechanism of ZNF139 in multi-drug resistance (MDR) of GC cells. MTT assay, RT-PCR, Western blotting were employed to detect susceptibility of GC cells to chemotherapeutic agents (5-FU, L-OHP) in vitro, and expressions of ZNF139 and MDR associated genes MDR1/P-gp, MRP1, Bcl-2, Bax were also detected. siRNA specific to ZNF139 was transfected into MKN28 cells, then chemosensitivity of GC cells as well as changes of ZNF139 and MDR associated genes were detected. It's found the inhibition rate of 5-FU, L-OHP to well-differentiated GC tissues and cell line was lower than that in the poorly differentiated tissues and cell line; expressions of ZNF139 and MDR1/P-gp, MRP1 and Bcl-2 in well-differentiated GC tissues and cell line MKN28 were higher, while Bax expression was lower. After ZNF139-siRNA was transfected into MKN28, ZNF139 expression in GC cells was inhibited by 90%; inhibition rate of 5-FU, L-OHP to tumor cells increased, and expressions of MDR1/P-gp, MRP1 and Bcl-2 were down-regulated, while Bax was up-regulated. ZNF139 was involved in GC MDR by promoting expressions of MDR1/P-gp, MRP1 and Bcl-2 and inhibiting Bax simultaneously.