[Application of copy number variation analysis based on raw data of next-generation sequencing in the molecular diagnosis for primary immunodeficiency disease].

Objective: To study the application of copy number variation (CNV) analysis based on the raw data of next-generation sequencing (NGS) in diagnosing primary immunodeficiency disease (PID). Methods: One hundred sixty-five patients with suspicious PID were tested by NGS in the Department of Rheumatology and Immunology, Shenzhen Children's Hospital during September 2014 and Mary 2017. The raw data of the patients who got negative result were further analyzed for the CNV with CNVkit software. The pathogenic CNV were identified in the databases including Resource of Asian Primary Immunodeficiency Diseases (RAPID), Human Gene Mutation Database (HGMD) and ClinVar with the known 344 pathogenic genes of PID. The associated literature from January 2010 to May 2019 were searched in Pubmed, Weip, Wanfang and CNKI database with key words as "primary immunodeficiency disease" "copy number variation" and "next generation sequencing" . Results: Ninety-five out of 165 patients (57.6%) had negative result of the NGS test, among whom the patients with immune dysregulation had the highest negative rate (68.6%, 24/35). CNV analysis found large fragment deletion in 12 patients, within which 7 was X-linked inheritance, 3 was autosomal recessive inheritance, 2 was autosomal dominant inheritance. Partial exon deletion was found in 4 patients while whole gene deletion in 8 patients. According to the review of literature, CNV was reported in 51 pathogenic genes of PID (14.8%, 51/344) , mainly intern deletion (70.6%, 36/51), while autosomal recessive inheritance (56.9%, 29/51) was the most common pattern. Conclusions: CNV is not rare in PID. When the phenotype is clear in the patients who have negative NGS test, CNV should be considered.

[Current status and prospects of integrated traditional Chinese and Western medicine for treatment of portal hypertension].

Portal hypertension refers to a series of clinical manifestations caused by elevated pressure of the portal vein system, which can cause portal hypertension by causing portal venous obstruction and / or increased blood flow. A typical clinical manifestation in patients with decompensated cirrhosis is portal hypertension. A severe complication of portal hypertension is esophagogastric varices bleeding, refractory ascites, and hepatic encephalopathy. The effective reduction of portal pressure can reduce the incidence of complications, improve the prognosis and reduce the mortality. At present, the commonly used clinical methods for reducing portal hypertension include drug therapy, minimally invasive interventions, surgical treatment, and liver transplantation. This article reviews the current status of integrated traditional Chinese and Western medicine for portal hypertension.

[The relationship between histone H3Ser10 phosphorylation and DNA damage in periphery blood lymphocytes of polycyclic aromatic hydrocarbons exposed workers].

Objective: To investigate the effect of polycyclic aromatic hydrocarbons (PAHs) exposure on the level of histone H3Ser10 phosphorylation (p-H3S10) and DNA damage degree in peripheral blood lymphocyte (PBLCs). Method: 75 coke oven workers from Benxi steel plant in Liaoning Province of China (PAHs-exposed group) and local 50 hot rolling workers (control group) were recruited in this study with age, working years, labor intensity and high temperature for matching factors using cluster sampling method in 2014. HPLC-fluorescence was performed to determine the level of urinary 1-hydroxypyrene (1-OHP), DNA damage and specific histone modification were measured in PBLCs of the subjects through comet assay and ELISA assay, respectively. Linear regression model analysis was used to analyze the differences among PAHs exposure, DNA damage and p-H3S10 level in two groups. The Mediation analysis was used to analyze the regulated relationships between urinary 1-OHP, DNA damage and histone modification through the bootstrap method. Results: Age of the control and the exposed group were (45.32±8.32) and (43.87±5.67) years old (P=0.284). The concentration of urinary 1-OHP, OTM value, Tail DNA% and p-H3S10 level in exposure group were higher than that in control group, while the M (P(5)-P(95)) of p-H3S10 levels in control and exposed group were 2.21 (0.68-4.71), 4.54 (1.85-23.91) (P<0.001). The degree p-H3S10 level was increased after the subgroups which were (2.59±1.19)%, (3.24±2.81)%, (5.55±3.25)%, (8.77±7.84)%, respectively, divided by quantitated 1-OHP concentration as P(0)-P(25), P(26)-P(50), P(51)-P(75) and P(76)-P(100) (P<0.001). We also found the correlations between urinary 1-OHP and p-H3S10 level or OTM value or Tail DNA%, ß (95%CI) were 0.264 (0.167-0.360), 0.500 (0.299-0.702), and 0.510 (0.384-0.671), respectively (P<0.001). Similar result was also observed between p-H3S10 level and OTM value or Tail DNA%, ß (95%CI) were 0.149 (0.073-0.226) and 0.220 (0.132-0.308) (P<0.001). Moreover, the mediation effect value of DNA damage on PAHs induced p-H3S10 alteration was 0.054(P=0.040). Conclusion: The results suggested that PAHs exposure could induce DNA damage and an increase in histone H3Ser10 phosphorylation in PBLCs. Particularly, the alteration of H3S10 phosphorylation may play an important role in regulating cell DNA damage repair.

[miR-34b-3p regulates the angiogenesis of senescent endothelial cell].

OBJECTIVE: To investigate the effect of miR-34b-3p on the proliferation, migration and tube formation of senescent endothelial cell. METHODS: Primary human umbilical vein endothelial cells (HUVECs) were cultured in vitro, and population doubling levels (PDLs) were calculated by passage. The young endothelial cell was defined as PDL8. The senescent endothelial cell was defined as PDL44. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) was applied to detect the expression of miR-34b-3p in PDL8 and PDL44 HUVECs. miR-34b-3p mimic and inhibitor were transfected into PDL8 and PDL44 HUVECs. Then, cell counting kit-8 (CCK-8), transwell and tube formation assays were used to determine the proliferation, migration and tube formation of HUVECs, respectively. RESULTS: miR-34b-3p was significantly up-regulated approximately 4.3 times in PDL44 HUVECs than that in PDL44 HUVECs (t=-4.528, P<0.05). The proliferation, migration, total tube length and branch points of miR-34b-3p in PDL8 HUVECs group were significantly higher approximately 1.2 (0.67/0.57), 1.2 (106/86), 1.4 (10 605/7 735) and 1.3 (41/31) times than that in PDL44 HUVECs group, respectively (t=3.237, 3.564, 5.165, 3.487, P<0.05 or P<0.01). Overexpression of miR-34b-3p had significantly inhibited proliferation, migration, total tube length and branch points approximately 2.2 (0.67/0.30), 2.3 (106/46), 1.6 (10 605/6 652) and 1.9 (41/22) times in PDL8 HUVECs, respectively (F=145.898, 53.026, 41.997, 36.341, all P<0.01). Repression of miR-34b-3p had significantly increased proliferation, migration, total tube length and branch points approximately 1.4 (0.77/0.57), 2.3 (198/86), 1.7 (13 073/7 735) and 2.3 (71/31) times in PDL44 HUVECs, respectively (F=14.815, 42.970, 167.063, 258.340, all P<0.01). CONCLUSION: The high expression of miR-34b-3p in senescent HUVECs could impair the proliferation, migration and tube formation of senescent endothelial cell.

ANP32B deficiency impairs proliferation and suppresses tumor progression by regulating AKT phosphorylation.

The acidic leucine-rich nuclear phosphoprotein 32B (ANP32B) is reported to impact normal development, with Anp32b-knockout mice exhibiting smaller size and premature aging. However, its cellular and molecular mechanisms, especially its potential roles in tumorigenesis, remain largely unclear. Here, we utilize 'knockout' models, RNAi silencing and clinical cohorts to more closely investigate the role of this enigmatic factor in cell proliferation and cancer phenotypes. We report that, compared with Anp32b wild-type (Anp32b(+/+)) littermates, a broad panel of tissues in Anp32b-deficient (Anp32b(-/-)) mice are demonstrated hypoplasia. Anp32b(-/-) mouse embryo fibroblast cell has a slower proliferation, even after oncogenic immortalization. ANP32B knockdown also significantly inhibits in vitro and in vivo growth of cancer cells by inducing G1 arrest. In line with this, ANP32B protein has higher expression in malignant tissues than adjacent normal tissues from a cohort of breast cancer patients, and its expression level positively correlates with their histopathological grades. Moreover, ANP32B deficiency downregulates AKT phosphorylation, which involves its regulating effect on cell growth. Collectively, our findings suggest that ANP32B is an oncogene and a potential therapeutic target for breast cancer treatment.

Distinguishing benign from malignant parotid gland tumours: low-dose multi-phasic CT protocol with 5-minute delay.

OBJECTIVES: To explore the percentage enhancement wash-out ratio (PEW) and relative PEW (RPEW) of low-dose multi-phasic computed tomography (CT) in distinguishing benign from malignant parotid gland tumours. METHODS: This study was approved by the ethics committee, and informed patient consent was obtained. 51 patients with parotid tumours proven by histopathology received CT, including 18 with pleomorphic adenomas, 14 with Warthin's tumours and 19 with malignant tumours. Size and attenuation of parotid tumours were measured. Compared with 5-min attenuation, the 30-s and 90-s PEW (PEW(30,) PEW(90)) and RPEW (RPEW(30), RPEW(90)) were calculated. RESULTS: There was a significant difference in PEW(30), RPEW(30), PEW(90) and RPEW(90) in the parotid neoplasms groups (P < 0.01), and statistical significance existed simultaneously in pleomorphic adenomas vs malignant tumours and Warthin's tumours vs malignant tumours according to SNK-q test. The optimal diagnosis results of malignancy with 100% specificity (32/32) was obtained by using a combination of the following criteria: -70% > PEW(30) < 36%, -30% > PEW(30) < 19%, PEW(90) > 12%, and the sensitivity (74%) for diagnosis of malignancy was yield. CONCLUSIONS: Wash-out ratio may assist in differentiating the benign from malignant parotid gland tumours. Combining the percentage of enhanced wash-out ratios of CT protocols can yield diagnostic results for malignancy.

α4 is highly expressed in carcinogen-transformed human cells and primary human cancers.

A regulator of the protein phosphatase 2A (PP2A), α4, has been implicated in a variety of functions that regulate many cellular processes. To explore the role of α4 in human cell transformation and tumorigenesis, we show that α4 is highly expressed in human cells transformed by chemical carcinogens including benzo(a)pyrene, aflatoxin B(1), N-methyl-N'-nitro-N-nitrosoguanidine, nickel sulfate and in several hepatic and lung cancer cell lines. In addition, overexpression of α4 was detected in 87.5% (74/80) of primary hepatocellular carcinomas, 84.0% (21/25) of primary lung cancers and 81.8% (9/11) of primary breast cancers, indicating that α4 is ubiquitously highly expressed in human cancer. Functional studies revealed that elevated α4 expression results in an increase in cell proliferation, promotion of cell survival and decreased PP2A-attributable activity. Importantly, ectopic expression of α4 permits non-transformed human embryonic kidney cells (HEKTER) and L02R cells to form tumors in immunodeficient mice. Furthermore, we show that the highly expressed α4 in transformed cells or human tumors is not regulated by DNA hypomethylation. A microRNA, miR-34b, that suppresses the expression of α4 through specific binding to the 3'-untranslated region of α4 is downregulated in transformed or human lung tumors. Taken together, these observations identify that α4 possesses an oncogenic function. Reduction of PP2A activity due to an enhanced α4-PP2A interaction contributes directly to chemical carcinogen-induced tumorigenesis.

Effect of intensity clamping on laser ablation by intense femtosecond laser pulses.

We have experimentally demonstrated that because of intensity clamping, when the laser peak power is higher than the critical power for self-focusing, further increase of the laser power cannot result in corresponding increase of the laser ablation rate of a metallic sample placed in gases. The ablation rate will finally approach a stabilized value. Also, the experimental technique implemented in our work could be potentially used to measure the self-focusing critical power and the nonlinear refractive index.

Activation of p42/44 mitogen-activated protein kinase pathway in long-term potentiation induced by nicotine in hippocampal CA1 region in rats.

AIM: To investigate the relationship between activation of p42/44 mitogen-activated protein kinase (MAPK) pathway and hippocampal long term potentiation (LTP) induced by nicotine in area CA 1. METHODS: Extracellular recording of population spike (PS) was performed within the pyramidal cell layer of hippocampal area CA1 in vitro; Western blot analysis was employed to detect the active phosphorylated state and the total protein expression of p42/44 MAPK. RESULTS: PD98059 concentration-dependently (25 micromol/L, 50 micromol/L) attenuated the induction of LTP induced by nicotine 10 micromol/L; both p42 and p44 MAPK were activated with their total protein expression increasing in CA1 subregion in response to LTP induced by nicotine. CONCLUSION: Activation of p42/44 MAPK pathway is required for hippocampal LTP induced by nicotine.

[Nitric oxide changes aortic function in rats with renal hypertension].

This work was undertaken to investigate the effect of nitric oxide (NO) on aortic function of two-kidney and one-clip (2K1C) rats with renal hypertension. Animals were divided into 5 groups: the sham operation, 2K1C, captopril, L-arginine and L-NAME groups. The results are as follows. At the 4th week after constriction of the left renal artery, the mean arterial pressure was significantly elevated. In isolated aortic rings, acetylcholine-induced dilation was attenuated, and phenylephrine induced contractile response was markedly enhanced. The level of aorta cGMP content was significantly lowered. These changes were abolished in 2K1C rats treated with captopril. L-arginine partially reversed the aortic vascular reactivity of 2K1C rats, and elevated aortic cGMP content. In 2K1C rats treated with nitric oxide synthase inhibitor, L-NAME, blood pressure was increased further, acetylcholine-induced aorta diastolic response was attenuated further and cGMP content reduced, while phenylephrine-induced contractile response was unaffected. These results suggest that deficiency of nitric oxide production and increase in renin-angiotensin system activity may contribute to vascular endothelial dysfunction of 2K1C rats, and these factors may be involved in development and maintenance of 2K1C renal hypertension.

Long-term potentiation induced by nicotine in CA1 region of hippocampal slice is Ca(2+)-dependent.

AIM: To observe the effects of Ca2+ on hippocampal long-term potentiation (LTP) induced by nicotine in CA1 region of rat hippocampal slice. METHODS: Extracellularly recorded population spikes (PS) of the pyramidal cell layer in the hippocampal CA1 region in vitro. RESULTS: Nicotine 1 mumol.L-1 induced LTP in the hippocampal CA1 region. It did not induce LTP in CA1 region when CA2+ was removed from artificial cerebrospinal fluid (ACSF). Nifedipine 1 and 10 mumol.L-1 partly inhibited LTP induced by nicotine, and thapsigargin 1 and 10 mumol.L-1 completely inhibited LTP induced by nicotine. CONCLUSION: LTP induced by nicotine in hippocampal CA1 region is Ca(2+)-dependent. Both Ca2+ influx and Ca2+ release participate in the induction of LTP.

[Effect of angiotensin II on c-fos expression and protein synthesis in cultured rat myocardial cells].

The present study was to investigate the effects of angiotensin II on c-fos mRNA expression and protein synthesis in cultured neonatal rat myocardial cells. The results showed that angiotensin II induced c-fos mRNA expression, increased protein content in a dose-dependent manner and stimulated 3H-leucine incorporation rate. All these effects were blocked by angiotensin II receptor antagonist saralasin. The angiotensin II-induced expression of c-fos gene was also blocked by Ca2+ channel antagonist nicardipine.

[Effect of ET-1 on intracellular free calcium in cultured neonatal myocardial cells].

In this present study, the effects of ET-1 on intracellular free calcium concentration ([Ca2+]i) and the underlying mechanisms were investigated in cultured neonatal rat myocardial cells loaded with fura-2/AM. The results are as follows. ET-1 induced an increase of [Ca2+]i in a dose-dependent manner, which consisted of a transient and sustained phase. BQ123, a selective ETA receptor antagonist, blocked the ET-1 induced [Ca2+]i responses, suggesting that these responses were mediated by ETA receptors. After removal of extracellular Ca2+, ET-1 induced the transient increase of [Ca2+]i without the sustained change. Protein kinase C (PKC) agonist PMA attenuated the ET-1 induced transient [Ca2+]i increase. Amiloride and nifedipine did not block the [Ca2+]i change induced by ET-1. After pretreatment of myocardial cells with pertussis toxin, ET-1 also induced the transient increase of [Ca2+]i but did not affect the sustained increase. These results suggest that the transient [Ca2+]i increase may involve pertussis toxin-insensitive G protein and the sustained one may be caused by extracellular calcium influx, in which pertussis toxin sensitive G protein is involved. Furthermore, PKC, but not Na+/H+ exchange, plays an important role in these effects.

[ET-1 induces the expression of prooncogene c-fos in cultured neonatal rat myocardial cells].

In the present study, the effect of endothelin-1 (ET-1) on the expression of proto-oncogene c-fos in cultured neonatal rat myocardial cells was investigated. The results are as follows: ET-1 induced c-fos expression in a dose-dependent manner. Selective ET(A) receptor antagonist blocked ET-1-induced responses. Protein kinase C(PKC) agonist PMA induced c-fos expression.PKC inhibitor staurosporine blocked ET-1 induced c-fos expression. Calcium channel blocker, nifedipine did not significantly affect the expression of c-fos induced by ET-1. These results suggest that in cultured neonatal cardiomyocytes, ET-1 induced c-fos gene expression is mediated by ET(A) receptor with the participation of protein kinase C, while the voltage-dependent L-type Ca(2+) channel is not involved.

[The role of G protein, protein kinase C and Na(+)-H+ exchanger in endothelin-1-induced cardiomyocyte hypertrophic responses].

Endothelin-1 (ET-1) has been shown to be a potent growth factor and to induce cardiac hypertrophy. In the present study, we examined the role of G protein, protein kinase C (PKC) and Na(+)-H+ exchanger in ET-1-induced cardiac hypertrophy in cultured neonatal rat cardiac myocytes. ET-1 (10(-10)-10(-7) mol/L) induced promotion of 3H-leucine incorporation, increase in cell protein content and cell surface area in a dose-dependent manner with EC50 value of 5.2 x 10(-10), 5.2 x 10(-10) and 7.3 x 10(-10) mol/L respectively. All of these ET-1-induced cardiomyocyte hypertrophic responses were completely blocked by pretreatment with staurosporine (2 nmol/L), a protein kinase C inhibitor, and stimulated by 4-phorbol, 12-myristate, 13-acetate (PMA) (10(-8)-10(-6) mol/L), a protein kinase C activator, in a dose-dependent manner. Pretreatment of amiloride (10(-4) mol/L), a Na(+)-H+ exchange inhibitor completely inhibited the ET-1-induced, but not PMA-induced cardiomyocyte hypertrophic responses. The ET-1-induced increase in cardiomyocyte protein synthesis and cell surface area was significantly inhibited by pretreatment with pertussis toxin (150 ng/ml). These results suggest that ET-1-induced cardiomyocyte hypertrophy was linked with pertussis toxin sensitive G protein, and PKC and Na(+)-H+ exchange may be an important intracellular signaling transduction pathway during ET-1-induced cardiac hypertrophy in cultured neonatal rat cardiac myocytes.

[Reduction of incidence of ischemia-reperfusion induced ventricular fibrillation by captopril].

To investigate the role of catecholamine and prostacyclin in ischemia reperfusion-induced ventricular fibrillation, experiments were performed in rat hearts using methods of radioimmunoassay and fluorohistochemistry. Regional myocardial ischemia was induced by ligation of the left coronary artery followed by reperfusion. In the ischemia reperfusion group, ventricular fibrillation during reperfusion took place in 78% of the hearts. In the group pretreated with captopril, the incidence of ventricular fibrillation decreased significantly (65.5%). In comparison with the ischemia reperfusion group, myocardial catecholamine content and 6-keto-PGF1 alpha of the captopril group were significantly increased (P < 0.01) while thromboxane B2 (TxB2) and TxB2/6-keto-PGF1 alpha were decreased (P < 0.01). In Ang II group, infusion of angiotensin II reversed the protective effect of captopril and restored the incidence of ventricular fibrillation (85%), while myocardial catecholamine content was not different from the ischemia reperfusion group (P > 0.05). Above results suggest that reduction of the incidence of ischemia reperfusion-induced ventricular fibrillation by captopril may be due to its inhibition on angiotensin II production with consequent reduction of the release of myocardial catecholamine, suppression of TxB2 and promotion of PGI2 synthesis.

Immunoglobulin E and circulating immune complexes in endolymphatic hydrops.

Levels of circulating immune complexes (CICs) in 59 patients with Meniere's disease and total serum immunoglobulin E (IgE) levels in a subgroup of 42 of the 59 were determined quantitatively for possible abnormalities of humoral immunity. Significant differences in average IgE levels between the 42 patients with Meniere's disease and 18 normal control subjects were not determined; however, five (11.9%) of the 42 patients were found to have obviously raised IgE levels. Elevated CIC levels were found in 19 (32.2%) of the 59 patients with Meniere's disease and in one (2.3%) of the 43 control subjects. This difference was statistically significant. The possible mechanisms of immune-mediated endolymphatic hydrops are discussed.

Human leukocyte antigens -A, -B, -C, and -DR and nasopharyngeal carcinoma in northern China.

We observed HLA associations in patients with nasopharyngeal carcinoma from northern China. There was an increased risk of nasopharyngeal carcinoma associated with HLA-B35 and a difference in the HLA association between patients with early- and late-onset disease. The frequency of B35 was significantly higher in patients than in control subjects, especially in early-onset patients (less than 30 years old). Late-onset patients had a higher frequency of DR2 as compared with normal subjects.