Single-cell and bulk RNA sequencing reveal heterogeneity and diagnostic markers in papillary thyroid carcinoma lymph-node metastasis.

PURPOSE: Papillary thyroid carcinoma (PTC) is characterized by lymph-node metastasis (LNM), which affects recurrence and prognosis. This study analyzed PTC LNM by single-cell RNA sequencing (scRNA-seq) data and bulk RNA sequencing (RNA-seq) to find diagnostic markers and therapeutic targets. METHODS: ScRNA-seq data were clustered and malignant cells were identified. Differentially expressed genes (DEGs) were identified in malignant cells of scRNA-seq and bulk RNA-seq, respectively. PTC LNM diagnostic model was constructed based on intersecting DEGs using glmnet package. Next, PTC samples from 66 patients were used to validate the two most significant genes in the diagnostic model, S100A2 and type 2 deiodinase (DIO2) by quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and immunohistochemical (IHC). Further, the inhibitory effect of DIO2 on PTC cells was verified by cell biology behavior, western blot, cell cycle analysis, 5-ethynyl-2'-deoxyuridine (EdU) assay, and xenograft tumors. RESULTS: Heterogeneity of PTC LNM was demonstrated by Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analysis. A total of 19 differential genes were used to construct the diagnostic model. S100A2 and DIO2 differ significantly at the RNA (p < 0.01) and protein level in LNM patient tissues (p < 0.001). And differed in PTC tissues with different pathologic typing (p < 0.001). Further, EdU (p < 0.001) and cell biology behavior revealed that PTC cells overexpressed DIO2 had reduced proliferative capacity. Cell cycle proteins were reduced and cells are more likely to be stuck in G2/M phase (p < 0.001). CONCLUSIONS: This study explored the heterogeneity of PTC LNM using scRNA-seq. By combining with bulk RNA-seq data, diagnostic markers were explored and the model was established. Clinical diagnostic efficacy of S100A2 and DIO2 was validated and the treatment potential of DIO2 was discovered.

[Application of multiplex PCR capillary electrophoresis and analysis of distribution characteristics of HPV infection subtypes].

Objective: To analyze the characteristics of human papillomavirus (HPV) infection and the distribution of HPV subtypes in Shijiazhuang, Hebei Province, and to explore the application evaluation of multiple PCR capillary electrophoresis fragment analysis for HPV typing test. Methods: A population-based cross-sectional study was conducted among 434 women (age range 17 to 74 years old, 260 patients and 174 physical examinations) included from May to August 2022 in Hebei General Hospital. HPV typing was detected by multiple PCR-capillary electrophoresis fragment analysis. Using the multiple fluorescence quantitative PCR kit as a reference, Chi-square test was used to analyze the diagnostic effect of multiple PCR-capillary electrophoresis fragment analysis, and the consistency was analyzed by Kappa value. Results: The total HPV infection rate was 45.85%(199/434), including 35.48% (154/434) of high-risk HPV (HR-HPV), 3.92% (17/434) of low-risk HPV (LR-HPV), 6.45% (28/434) of HR-HPV and LR-HPV mixed infection, 27.88% (121/434) of single type HPV and 17.97% (78/434) of multi type HPV. HPV52 (9.68%, 42/434), HPV16 (6.91%, 30/434), and HPV58 (6.91%, 30/434) are common HPV subtypes. The positive rate of physical examination was 45.40% (79/174), which was slightly lower than that of patients 46.15% (120/260), there was no significant difference (χ2=0.024,P>0.05). The highest infection rate in the 17-30 age group was 54.76% (46/84), and there was no statistical difference among the age groups(χ2=4.123,P>0.05). The sensitivity and specificity of multiplex PCR capillary electrophoresis fragment analysis were 92.96% and 94.04%, respectively, and Kappa value was 0.870, with the multiplex fluorescent quantitative PCR as the reference. Conclusion: HPV infection may appear younger, and the positive rate of HR-HPV infection is the highest, with HPV52, 16, 58 as the main infection subtypes. The detection results of multiplex PCR capillary electrophoresis fragment analysis method are highly consistent with those of multiplex fluorescent quantitative PCR method, which is suitable for HPV DNA typing.

[Safety and efficacy of dual guiding catheter kissing technique in the treatment of stent partially dislodgement in coronary artery].

Objective: To investigate the safety and efficacy of dual guiding catheter kissing technique (DCK) in the treatment of stent partly dislodgement in coronary artery. Methods: The study retrospectively involved 6 hospitalized patients with coronary artery stent partly dislodgement during PCI at The First Affiliated Hospital of Zhengzhou University from February 2016 to June 2019, DCK was used in these patients. We observe the success rate of stent retrieval, success rate of PCI, incidence of complications and major adverse cardiovascular events in 1 year follow up. Results: 6 patients were involved, of which 3 are male, ages range 49 to 68 years old, 4 patients are diagnosed with unstable angina, the other two are stable angina. All the partially disloged stents in the 6 patients were successfully removed from coronary artery. Except for 1 patient who refused coronary artery stenting again, the other 5 patients were successfully implanted coronary artery stenting. No serious complications occurred, no patients died and no major adverse cardiovascular events happened during 1 year follow up. Conclusions: DCK is safe and effective to remove partially dislodged stent in coronary artery.

[Determination of phenyl glycidyl ether in workplace air by solvent desorption gas chromatography].

Objective: To establish a solvent desorption gas chromatographymethod for the determination of phenyl glycidyl ether (PGE) . Methods: From October to December 2020, PGE in the air of workplace was collected with carbon tube and desorbed by 25% acetone-carbon disulfide. The target toxicant was separated with the gas chromatography (GC) column and analyzed with flame ionization detector (FID), and quantified by peak area. Results: The linear range of PGE in the air of workplace was 10.0-1109.0 µg/ml, the linear equation was y=1.156x-4.328, with a correlation coefficient of 0.9997. The limit of detection was 3.0 µg/ml. The lower limit of quantification was 10.0 µg/ml. The intar-batch and inter-batch precisionswere 4.9%-6.4% and 6.2%-6.9%, respectively. The recovery rate was ranged from 97.2%-98.8%, the average collection efficiency was 100%, and the average extraction efficiency was 90.1%. The samples could be stored at 4 ℃ for 7 d. Conclusion: This method has high precision and good accuracy, and it is applicable for the determination of PGE in workplace air.

[Long-term efficacy and safety of simultaneous integrated boost radiotherapy in non-operative esophageal squamous cell carcinoma: a multicenter retrospective data analysis (3JECROG R-05)].

Objective: To analyze the survival benefits and treatment related toxic effects of simultaneous integrated boost intensity-modulated radiotherapy (SIB-RT) for non-operative esophageal squamous cell carcinoma patients. Methods: The data of 2 132 ESCC patients who were not suitable for surgery or rejected operation, and underwent radical radiotherapy from 2002 to 2016 in 10 hospitals of Jing-Jin-Ji Esophageal and Esophagogastric Cancer Radiotherapy Oncology Group (3JECROG) were analyzed. Among them, 518 (24.3%) cases underwent SIB (SIB group) and 1 614 (75.7%) cases did not receive SIB (No-SIB group). The two groups were matched with 1∶2 according to propensity score matching (PSM) method (caliper value=0.02). After PSM, 515 patients in SIB group and 977 patients in No-SIB group were enrolled. Prognosis and treatment related adverse effects of these two groups were compared and the independent prognostic factor were analyzed. Results: The median follow-up time was 61.7 months. Prior to PSM, the 1-, 3-, and 5-years overall survival (OS) rates of SIB group were 72.2%, 42.8%, 35.5%, while of No-SIB group were 74.3%, 41.4%, 31.9%, respectively (P=0.549). After PSM, the 1-, 3-, and 5-years OS rates of the two groups were 72.5%, 43.4%, 36.4% and 75.3%, 41.7%, 31.6%, respectively (P=0.690). The univariate survival analysis of samples after PSM showed that the lesion location, length, T stage, N stage, TNM stage, simultaneous chemoradiotherapy, gross tumor volume (GTV) and underwent SIB-RT or not were significantly associated with the prognosis of advanced esophageal carcinoma patients who underwent radical radiotherapy (P<0.05). Cox model multivariate regression analysis showed lesion location, TNM stage, GTV and simultaneous chemoradiotherapy were independent prognostic factors of advanced esophageal carcinoma patients who underwent radical radiotherapy (P<0.05). Stratified analysis showed that, in the patients whose GTV volume≤50 cm(3), the median survival time of SIB and No-SIB group was 34.7 and 30.3 months (P=0.155), respectively. In the patients whose GTV volume>50 cm(3), the median survival time of SIB and No-SIB group was 16.1 and 20.1 months (P=0.218). The incidence of radiation esophagitis and radiation pneumonitis above Grade 3 in SIB group were 4.3% and 2.5%, significantly lower than 13.1% and 11% of No-SIB group (P<0.001). Conclusions: The survival benefit of SIB-RT in patients with locally advanced esophageal carcinoma is not inferior to non-SIB-RT, but without more adverse reactions, and shortens the treatment time. SIB-RT can be used as one option of the radical radiotherapy for locally advanced esophageal cancer.

[Prognostic analysis of definitive radiotherapy for early esophageal carcinoma(T1-2N0M0): a multi-center retrospective study of Jing-Jin-ji Esophageal and Esophagogastric Cancer Radiotherapy Oncology Group].

Objective: To evaluate the prognostic factors of T1-2N0M0 esophageal squamous cell carcinoma (ESCC) treated with definitive radiotherapy. Methods: The clinical data of 196 patients with T1-2N0M0 ESCC who were treated with definitive radiotherapy in 10 hospitals were retrospectively analyzed. All sites were members of Jing-Jin-Ji Esophageal and Esophagogastric Cancer Radiotherapy Oncology Group (3JECROG). Radiochemotherapy were applied to 78 patients, while the other 118 patients received radiotherapy only. 96 patients were treated with three-dimensional conformal radiotherapy (3DCRT) and 100 treated with intensity-modulated radiotherapy (IMRT). The median dose of plan target volume(PTV) and gross target volume(GTV) were both 60 Gy. The median follow-up time was 59.2 months. Log rank test and Cox regression analysis were used for univariat and multivariate analysis, respectively. Results: The percentage of normal lung receiving at least 20 Gy (V(20)) was (18.65±7.20)%, with average dose of (10.81±42.05) Gy. The percentage of normal heart receiving at least 30 Gy (V(30)) was (14.21±12.28)%. The maximum dose of exposure in spinal cord was (39.65±8.13) Gy. The incidence of radiation pneumonia and radiation esophagitis were 14.80%(29/196) and 65.82%(129/196), respectively. The adverse events were mostly grade 1-2, without grade 4 toxicity. Median overall survival (OS) and progression-free survival (PFS) were 70.1 months and 62.3 months, respectively. The 1-, 3- and 5-year OS rates of all patients were 75.1%、57.4% and 53.2%, respectively. The 1-, 3- and 5-year PFS rates were 75.1%、57.4% and 53.2%, respectively. Multivariate analysis demonstrated that patients'age (HR=1.023, P=0.038) and tumor diameter (HR=1.243, P=0.028)were the independent prognostic factors for OS, while tumor volume were the independent prognostic factor for PFS. Conclusions: Definitive radiotherapy is a promising therapeutic method in patients with T1-2N0M0 ESCC. Patients' age, tumor diameter and tumor volume may impact patients' prognosis.

[Efficacy analysis of the radiotherapy and chemotherapy in patients with stage â £ esophageal squamous carcinoma: a multicenter retrospective study of Jing-Jin-Ji Esophageal and Esophagogastric Cancer Radiotherapy Oncology Group (3JECROG R-01F)].

Objective: To evaluate the survival and prognostic factors of radiotherapy in patient with Ⅳ stage esophageal squamous carcinoma treated with radiation or chemoradiation. Methods: The medical records of 608 patients with stage Ⅳ esophageal squamous cell carcinoma who met the inclusion criteria in 10 medical centers in China from 2002 to 2016 were retrospectively analyzed. The overall survival and prognostic factors of all patients at 1, 3 and 5 years were analyzed. Results: The 1-, 3-, 5- year overall survival (OS) rates was 66.7%, 29.5% and 24.3% in stage ⅣA patients, and 58.8%, 29.0% and 23.5% in stage ⅣB patients. There was no statistical difference between the two groups (P=0.255). Univariate analysis demonstrated that the length of lesion, treatment plan, planned tumor target volume (PGTV) dose, subsequent chemotherapy, and degrees of anemia, radiation esophagitis, radiation pneumonia were related to the prognoses of patients with Ⅳ stage esophageal carcinomas after radiotherapy and chemotherapy (P<0.05). Multivariate analysis demonstrated that PGTV dose (OR=0.693, P=0.004), radiation esophagitis (OR=0.867, P=0.038), and radiation pneumonia (OR=1.181, P=0.004) were independent prognostic factors for OS. Conclusions: For patients with stage Ⅳ esophageal squamous cell carcinoma, chemoradiotherapy followed by sequential chemotherapy is recommended, which can extend the total survival and improve the prognosis of the patients. PGTV dose more than 60 Gy has better efficacy.

[Study on safety of adjuvant radiotherapy concurrent with weekly chemotherapy for stage â ¡B-â £A esophageal carcinoma after radical resection].

Objective: To evaluate the tolerability and short-term efficacy of chemo-radiotherapy in 125 patients with stage ⅡB-ⅣA esophageal carcinoma after radical resection. Methods: We retrospectively evaluated the rate of completion, toxicity and survival of patients undergoing adjuvant concurrent chemo-radiotherapy after radical resection of esophageal carcinoma from January 2004 to December 2014 in our institution. The survival rate was determined by the Kaplan-Meier method and analyzed using the log-rank test. Multivariate prognostic analysis was performed using the Cox proportional hazard model. Results: 122 patients received more than 50 Gy dose (97.6%). A total of 52 patients received more than 5 weeks chemo-radiotherapy (41.6%), while 73 patients underwent only 1-4 weeks (58.4%). The median following up was 48.4 months. 8 patients lost follow up (6.4%). The 1-year and 3-year overall survival rate were 91.6% and 57.0%, respectively, with a median survival time of 64.4 months. The 1-year and 3-year disease free survival rate were 73.2% and 54.3%, respectively, with a median disease free survival time of 59.1 months. The most common acute complications associated with chemo-radiotherapy were myelosuppression, radiation esophagitis and radiation dermatitis, the majority of which were Grade 1-2. Of the 125 patients, there were 59 cases of recurrence, including 23 cases with local regional recurrence, 26 cases with hematogenous metastasis, and 8 cases with mixed recurrence. Univariate analysis showed that the numbers of concurrent chemotherapy was associated with the overall survival (P=0.006). But receiving more than 5 weeks was not the prognostic factor compared to 1 to 4 weeks chemotherapy (P=0.231). Multivariate analysis showed that only the numbers of concurrent chemotherapy was an independent prognostic factor (P=0.010). Conclusions: Postoperative radiotherapy concurrent with weekly chemotherapy could improve the overall survival and decrease the recurrence for stage ⅡB-ⅣA esophageal carcinoma after radical resection. However, the completion rate of chemotherapy was low, so it was necessary to explore reasonable regimens to improve the completion rate and carry out prospective randomized controlled trial.

[Simultaneous treatments in patients with severe carotid artery stenosis and coronary artery disease].

Objective: To implement simultaneous treatments in patients with severe carotid artery stenosis and coronary artery disease (CAD), we sought to investigate its efficacy of reducing perioperative major adverse cardiac and cerebrovascular event (MACCE). Methods: Brain-and-Heart treatment team in Peking University International Hospital performed hybrid Digital Substraction Angiography (DSA) of carotid artery and coronary artery for 37 patients meeting the group criterion from September 2017 to February 2019.Twelve patients were diagnosed and received simultaneous treatments of severe carotid artery stenosis and coronary artery disease after hybrid DSA. We conducted the retrospective study and made analysis of these patients. Results: The diagnosis rate by simultaneous treatmentsin patients with carotid artery stenosis and coronary artery disease is 59.5% (22/37), the rate of severe carotid artery stenosis or multi-vessel CAD is 77.3% (17/22).The rate of severe carotid artery stenosis with CAD is 54.5% (12/22), therate of simultaneous treatmentsis 83.3%(10/12). Nine patients post-operative symptoms release, one patient with ischemic stroke after CABG, and two patients of medical therapy with stable symptoms. Conclusion: Simultaneous treatments in patients with severe carotid artery stenosis and coronary artery disease not only reveal the positive correlation between carotid stenosis and CAD, but also accurately evaluate severity degree or rapidly formulate scheme and reduce MACCE.

[Application of combined angiography in diagnosis and treatment of carotid artery stenosis].

Objective: To evaluate the safety and clinical value of combined cardiac and cerebral angiography in the diagnosis and treatment of patients with carotid artery stenosis. Methods: Clinical data of patients from May 2016 to June 2019 were collected. The patient underwent combined angiography under local anesthesia. Clinical characteristics, prognosisand complications were retrospectively analyzed. Results: A total of 45 patients with carotid artery stenosis received combined angiography, among which 12 patients had no obvious carotid artery stenosis. There were 22 cases with unilateral carotid artery stenosis, including 12 cases with mild stenosis and 10 cases with moderate to severe stenosis. There were 11 cases of bilateral stenosis, including 3 cases with mild stenosis and 8 cases with moderate to severe stenosis. 12 patients had no obvious stenosis incoronary arteries, 11 patients had single-vessel disease, 7 patients had 2-vessel disease, and 15 patients had 3-vessel disease. In the whole group of patients, surgical indications are available of 18 patients with carotid artery stenosis, 4 of the mreceived combined therapy. 2 of them under went Carotid Endarterectomy (CEA)+Percutaneous Transluminal Coronary Intervention (PCI), 2 under went Carotid Artery Stenting (CAS)+PCI. CAS+coronary artery angiography was performed in 3 cases. CEA+coronary angiography was performed in 5 cases. 5 patients received conservative treatment after combined angiography. In one case, the condition of coronary artery was so complicated that PCI was performed firstly. No cardiac or cerebrovascular complications such as myocardial infarction or cerebral infarction occurred during perioperative period. Conclusion: Combined cardio-cerebrovascular angiography is a good method to evaluate the cardiovascular and cerebrovascular in the diagnosis and treatment of patients with carotid artery stenosis, which can provide a clear diagnosis for treatment with a low complications frequency.

[Clinical significance of NS1-BP expression in esophageal squamous cell carcinoma].

Objective: To investigate the clinical significance of NS1-BP expression in patients with esophageal squamous cell carcinoma (ESCC), and to study the roles of NS1-BP in proliferation and apoptosis of ESCC cells. Methods: A total of 98 tumor tissues and 30 adjacent normal tissues from 98 ESCC patients were used as study group and control group, and these samples were collected in Sun Yat-Sen University Cancer Center between 2002 and 2008. In addition, 46 ESCC tissues which were collected in Cancer Institute and Hospital of Tianjin Medical University were used as validation group. Expression of mucosal NS1-BP was detected by immunohistochemistry. Kaplan-Meier curve and log-rank test were used to analyze the survival rate. Multivariate Cox proportional hazard model was used to analyze the prognostic factors. Furthermore, NS1-BP was over expressed or knocked down in ESCC cells by transient transfection. Protein levels of c-Myc were detected by western blot. Cell viability and apoptosis was analyzed by MTT assay and flow cytometry. Results: Among all of tested samples, NS1-BP were down-regulated in 9 out of 30 non-tumorous normal esophageal tissues (30.0%) and 85 out of 144 ESCC tissues (59.0%), respectively, showing a statistically significant difference (P=0.012). In the study group, three-year disease-free survival rate of NS1-BP high expression group (53.2%) was significantly higher than that of NS1-BP low expression group (27.6%; P=0.009). In the validation group, the three-year disease-free survival rates were 57.8% and 25.5% in NS1-BP high and low levels groups, respectively, showing a similar results (P=0.016). Importantly, multivariate analyses showed that low expression of NS1-BP was an independent predictor for chemoradiotherapy sensitivity and shorter disease-free survival time in ESCC patients(P<0.05 for all). Furthermore, overexpressed NS1-BP in TE-1 cells repressed c-Myc expression, inhibited cell proliferation and promoted apoptosis. In contrast, knockdown NS1-BP in KYSE510 cells induced c-Myc expression, increased cell proliferation and repressed apoptosis. Conclusions: NS1-BP is an independent favorable prognostic factor in ESCC. It inhibits cell proliferation and enhances cell apoptosis via repressing c-Myc. Targeting NS1-BP may be a new therapeutic strategy for ESCC patients.

[Clinical experience and next-generation sequencing analysis of encephalitis caused by pseudorabies virus].

Objective: To detect potential pathogens including pseudorabies virus in patients with encephalitis of unknown etiology in China and describe novel encephalitic entities. Methods: Patients with clinically suspected infectious encephalitis were enrolled in a multicenter study to identify the pathogens in PUMCH Encephalitis Program.Next-generation sequencing(NGS) of cerebrospinal fluid (CSF) was used in patients with encephalitis of unknown etiology enrolled from 2016 to 2017.The patients diagnosed as PRV encephalitis were studied to describe this novel entity. Results: The four patients(3 male, 1 male, 38-54 years old) had occupational exposure to raw park when working in the production or marketing of pork and at least one got injured during pork-cutting.Two of them were confirmed with NGS of CSF, and anti-PRV antibodies were positive in 3 patients whose serum was available for serological analysis.They all presented with an acute onset of fever, convulsion, loss of consciousness and respiratory failure within 1 to 4 days and rapidly deteriorated even on extensive treatment.All the patients needed ICU admission and 3 needed mechanical ventilation.Two patients also had bilateral retinitis.Neuroimaging revealed symmetric gray matter lesions including limbic system, basal ganglia and midbrain without obvious hemorrhage.Lumbar puncture revealed elevated intracranial pressure and lymphocytic pleocytosis [(6-64)×10(6)/L] of CSF.The patients failed to response to the treatment of acyclovir combined with intravenous immunoglobulin and steroids.Modified Rankin Score was 3, 5, 5 and 6 (died) for the 4 patients respectively on last follow-up. Conclusions: PRV could be a cause of severe encephalitis.The patients with suspected pseudorabies encephalitis (PRE) need to be tested for PRV DNA timely.Severe encephalitis with bilateral involvement of limbic system, basal ganglion, thalamus and midbrain in patient with occupational exposure indicate this emerging infectious encephalitis.

[The optimal cutoff value of waist-to-height ratio in Chinese: based on cardiovascular risk and metabolic disease].

Objective: Waist-to-height ratio (WHtR), a measurement of the distribution of body fat, correlated with abdominal obesity indicating that it might be a better predictor of cardiovascular risk and metabolic disease. We, therefore, evaluated optimal WHtR cutoff points according to the risk of framingham risk score (FRS) and metabolic syndrome (MS) in Chinese. Methods: The subjects were from China National Diabetes and Metabolic Disorders Survey during 2007-2008. Receiver operating characteristic analysis was used to examine the optimal cutoff values of WHtR according to the risk of FRS and MS. Results: A total of 27 820 women and 18 419 men were included in the evaluation. The average age was (45.0±13.7) years. The proportions of FRS ≥10% and MS increased with WHtR both in men and women. The cutoff points of WHtR for the risk of FRS ≥10% and MS were 0.51, 0.52 in men, and 0.52, 0.53 in women, respectively. When FRS ≥10% and MS were taken into consideration with a certain weights, the pooled cutoffs of WHtR were 0.51 in men, and 0.53 in women, respectively. By using the similar method, the optimized cutoff points were 0.52, 0.51, 0.50 for men and 0.51, 0.53, 0.54 for women in age group 20-39, 40-59 and ≥60 years, respectively. Conclusions: The optimal cutoffs of WHtR are 0.51 in men, and 0.53 in women for FRS≥10% in combination with MS indicating that this WHtR cutoff points might be used as indexes to evaluate obesity and risk of obesity-related diseases.

Effects of donor cells' sex on nuclear transfer efficiency and telomere lengths of cloned goats.

The aim of this study was to investigate the effects of donor cells' sex on nuclear transfer efficiency and telomere length of cloned goats from adult skin fibroblast cells. The telomere length of somatic cell cloned goats and their offspring was determined by measuring their mean terminal restriction fragment (TRF) length. The result showed that (i) reconstructed embryos with fibroblast cells from males Boer goats obtained significantly higher kids rate and rate of live kids than those of female embryos and (ii) the telomere lengths of four female cloned goats were shorter compared to their donor cells, but five male cloned goats had the same telomere length with their donor cells, mainly due to great variation existed among them. The offspring from female cloned goats had the same telomere length with their age-matched counterparts. In conclusion, the donor cells' sex had significant effects on nuclear transfer efficiency and telomere lengths of cloned goats.

GENOME-WIDE MAPPING OF COPY NUMBER VARIATIONS IN COMMERCIAL HYBRID PIGS USING A HIGH-DENSITY SNP GENOTYPING ARRAY.

Copy number variations (CNVs) are important forms of structural variation in human and animals and can be considered as a major genetic component of phenotypic diversity. Here we used the Illumina PorcineSNP60 BeadChip V2 and a DLY [Duroc x (Large White x Landrace)] commercial hybrid population to identify 272 CNVs belonging to 165 CNV regions (CNVRs), of which 66 are new. As CNVRs are specific to origin of population, our DLY-specific data is an important complementary to the existing CNV map in the pig genome. Eight CNVRs were selected. for validation by quantitative real-time PCR (qRT-PCR) and the accurate rate was high (87.25%). Gene function analysis suggested that a common CNVR may play an important role in multiple traits, including growth rate and carcass quality.

[Icariin reduces S-nitrosogultathione induced endothelial cell apoptosis through modulating AKT/P53 pathway].

OBJECTIVE: To observe the effects of icariin on S-nitrosogultathione(GSNO) induced endothelial cell apoptosis, and to explore the relative mechanisms. METHODS: EA.hy926 cell line was provided by Zhejiang University and cells were divided into blank control group, GSNO group (1 mmol/L GSNO), icariin (ICA) intervention group (GSNO+ different concentrations (high, medium and low: 10, 1 and 0.1 µmol/L ICA) and 1 µmol/L LY294002 pretreatment groups (AKT protease pathway inhibitor on top of ICA groups) by cluster random sample method. After 48 hours. EA.hy 926 cell survival was detected by thiazolyl blue tetrazolium bromicle (MTT) method. Lactate dehydrogenase (LDH) activity, malonaldehyde (MDA) content, reactive oxygen species (ROS) level, mitochondrial membrane potential were also measured. The protein expression of protein kinase B(AKT)/phosphorylation protein kinase B(p-AKT), people tumor-suppressor protein (protein 53, P53), cytochrome C (CYC), endothelial nitric oxide synthetase (eNOS)/phosphorylation endothelial nitric oxide synthetase (p-eNOS), procaspase-3/caspase-3 was detected by Western blot. RESULTS: (1) The cell survival rate was significantly lower in GSNO group than in the blank control group (P< 0.01), which was significantly higher in the high, medium and low concentration ICA groups than in the GSNO group (all P< 0.01). (2) The LDH activity was significantly higher in the GSNO group than in the blank control group ((142.65±5.56) U/L vs. (50.01±3.42) U/L, P< 0.05), which was significantly reduced by high, medium and low concentration ICA ((98.02±3.52), (105.29±6.89) and (117.16±4.27) U/L vs. (142.65±5.56) U/L, all P< 0.05) in a dose-dependent manner. (3) The MDA content was significantly higher in GSNO group than in the blank control group ((11.14±0.37) nmol/mg vs. (5.21±0.18) nmol/mg, P< 0.05), which could be reduced by pretreatment with high, medium and low concentration ICA ((6.60±0.41), (6.83±0.21) and (8.29±0.07) nmol/mg vs. (11.14 ±0.37) nmol/mg, all P< 0.05). (4) The ROS content was significantly higher in GSNO group than in the blank control group ((173.15±11.12)% (relative ratio to the blank control group), P< 0.05), which could be significantly reduced by pretreatment with high, medium and low concentration ICA ((122.56±8.09)%, (134.52±9.09)%, and(149.89±9.16)% (the ratio of the above are compared with the blank control group), P< 0.05). (5) The mitochondrial membrane potential was significantly higher in the GSNO group (0.84 ± 0.04) than in the blank control group (0.12 ±0.12), which could be significantly reduced by pretreatment with high, medium and low concentration ICA ((0.57±0.08), (0.63±0.02), (0.66±0.04) vs. (0.84±0.04), all P<0.05). (6) The expression of AKT/p-AKT was significantly lower in GSNO group than in the blank control group (P< 0.05), which could be significantly upregulated by pretreatment with high, medium and low concentration ICA in a concentration-dependent manner, above effects could be blocked by LY294002 (all P<0.05). (7) The expression of P53 was significantly higher in GSNO group than in the blank control group (P< 0.05), which could be significantly down regulated by pretreatment with high, medium and low concentration ICA in a concentration-dependent manner, above effects could be blocked by LY294002(all P<0.05). (8) The expression of CYC and caspase-3 was significantly reduced in the mitochondria and increased in cytoplasm post GSNO treatment compared to blank control group, which could be reversed by pretreatment with high, medium and low concentration ICA(all P<0.05). (9) The expression of eNOS/p-eNOS was similar between GSNO and the blank control group, while it was significantly upregulated by pretreatment with high, medium and low concentration ICA and this effect could be blocked by LY294002(all P<0.05). CONCLUSION: Icariin could reduce GSNO induced endothelial cell apoptosis through activating AKT pathway and downregulating P53 activity.

[Association between hematocrit and risk of incident hypertension: a cohort study].

Objective: To explore the association between hematocrit level and risk of incident hypertension. Method: Subjects who participated at least two times routine health check-up in Health Management Center of Shandong Province Hospital between January 2005 and January 2010 were eligible for inclusion. After excluding participants with known hypertension and other related diseases, a prospective cohort with 20 606 subjects (female: 8 218, male: 12 388) was established. Cox-proportional hazard model was used to assess the association between hematocrit and the development of hypertension for female and male respectively. Results: During the 51 352 person-years of follow-up, newly developed hypertension was confirmed in 3 695 cases. For female, the age-adjusted hazard ratios (95%CI) for incident hypertension were 1.00 (reference), 1.06(0.86-1.29), 1.37(1.14-1.65), 1.60(1.34-1.92), respectively (P for trend<0.000 1) through the 3 quartiles of hematocrit levels.After adjusting multiple factors (age, smoking, drinking habit, physical activity, body mass index(BMI), systolic blood pressure, fasting blood glucose, serum creatinine, high-density lipoprotein, gamma-glutamyl transferase, white blood count), the hazard ratios (95%CI) were 1.00(reference), 1.05(0.85-1.29), 1.25(1.03-1.51), and 1.22(1.00-1.48), respectively (P for trend=0.016 9). For male, the hazard ratio (the highest vs. the lowest hematocrit level) after adjusting age or age and life style factors (smoking, drinking habit, physical activity) was 1.23 (1.11-1.37), 1.21(1.09-1.35), respectively. Other analyses of relationship hematocrit with incident hypertension were not statistically significant in male. Conclusions: Higher hematocrit level is associated with higher risk of incident hypertension, especially in female.

Effects of humic acid and ionic strength on TiO2 nanoparticles sublethal toxicity to zebrafish.

The stability and bioavailability of titanium dioxide nanoparticles (TiO2 NPs) suspension could be modified by the physicochemical properties of solution. In the present study, the effect of humic acid (HA) and ionic strength (by adding NaCl) on aggregation and sedimentation of TiO2 NPs suspension were investigated. Accordingly, the sublethal toxicity of TiO2 NPs suspensions with different HA and NaCl concentrations toward zebrafish (Danio rerio) was evaluated by monitoring the changes of superoxide dismutase, catalase, malonaldehyde and glutathione in gill, liver and intestine. The results showed that the aggregations formation and hydrodynamic diameter of TiO2 NPs in suspensions are not essential characteristics to decide toxicity. The varied oxidative stress responses detected in gill, liver and intestine derived from different toxicity mechanisms of TiO2 NPs. Nevertheless, the oxidative stress could be suppressed by the adding of HA and/or the increase of ionic strength, which can decrease the bioavailability of TiO2 NPs in water. The study suggests that the environmental factors, such as HA and ionic strength, are important for the fate (aggregation and sedimentation) and toxicity of nanomaterials in aquatic environment.

Regulating effect of MMP-9 and TIMP-1 in pituitary adenoma invasion.

Pituitary adenomas can cause endocrine disorder and organ damage, with some aggressive ones leading to a high postoperative recurrence rate. The occurrence and development of these type of tumors is closely related with matrix metalloproteinases (MMPs) and endogenous specific tissue inhibitor of MMPs (TIMPs). In this study, the relationship between pituitary adenoma invasion and the changes in MMP-8 and TIMP-1 expressions is analyzed. Specimens from sixty patients with pituitary adenoma were collected in our hospital after surgery, including thirty cases of invasive pituitary adenomas and thirty cases of noninvasive pituitary adenomas. Western blotting and real-time PCR were used to detect MMP-8/TIMP-1 protein and mRNA levels, respectively, in the two types of pituitary adenomas, while ELISA was used to detect both compounds' levels in the patient's serum. Compared with noninvasive pituitary adenomas, MMP-8 was significantly overexpressed in invasive pituitary adenomas, while TIMP-1 was obviously lower (P < 0.05 for both). Moreover, MMP-8 mRNA expression in invasive pituitary adenomas was significantly higher than in noninvasive pituitary adenomas, while TIMP-1 mRNA expression was markedly lower (P < 0.05 for both). Finally, MMP-8 expression in the serum is upregulated in patients with invasive pituitary adenomas relative to the noninvasive ones, and the expression of TIMP-1 significantly reduced (P < 0.05 for both). These results show that increased MMP-8 and decreased TIMP-1 expressions are closely related to the invasive pituitary adenoma, and can be helpful for the evaluation.

SUSCEPTIBILITY LOCI FOR UMBILICAL HERNIA IN SWINE DETECTED BY GENOME-WIDE ASSOCIATION.

Umbilical hernia (UH) is a complex disorder caused by both genetic and environmental factors. UH brings animal welfare problems and severe economic loss to the pig industry. Until now, the genetic basis of UH is poorly understood. The high-density 60K porcine SNP array enables the rapid application of genome-wide association study (GWAS) to identify genetic loci for phenotypic traits at genome wide scale in pigs. The objective of this research was to identify susceptibility loci for swine umbilical hernia using the GWAS approach. We genotyped 478 piglets from 142 families representing three Western commercial breeds with the Illumina PorcineSNP60 BeadChip. Then significant SNPs were detected by GWAS using ROADTRIPS (Robust Association-Detection Test for Related Individuals with Population Substructure) software base on a Bonferroni corrected threshold (P = 1.67E-06) or suggestive threshold (P = 3.34E-05) and false discovery rate (FDR = 0.05). After quality control, 29,924 qualified SNPs and 472 piglets were used for GWAS. Two suggestive loci predisposing to pig UH were identified at 44.25MB on SSC2 (rs81358018, P = 3.34E-06, FDR = 0.049933) and at 45.90MB on SSC17 (rs81479278, P = 3.30E-06, FDR = 0.049933) in Duroc population, respectively. And no SNP was detected to be associated with pig UH at significant level in neither Landrace nor Large White population. Furthermore, we carried out a meta-analysis in the combined pure-breed population containing all the 472 piglets. rs81479278 (P = 1.16E-06, FDR = 0.022475) was identified to associate with pig UH at genome-wide significant level. SRC was characterized as plausible candidate gene for susceptibility to pig UH according to its genomic position and biological functions. To our knowledge, this study gives the first description of GWAS identifying susceptibility loci for umbilical hernia in pigs. Our findings provide deeper insights to the genetic architecture of umbilical hernia in pigs.