Multiplex fluorescent amplification-refractory mutation system PCR method for the detection of 10 genetic defects in Holstein cattle and its comparison with the KASP genotyping assay.

The common deleterious genetic defects in Holstein cattle include haplotypes 1-6 (HH1-HH6), haplotypes for cholesterol deficiency (HCD), bovine leukocyte adhesion deficiency (BLAD), complex vertebral malformation (CVM) and brachyspina syndrome (BS). Recessive inheritance patterns of these genetic defects permit the carriers to function normally, but homozygous recessive genotypes cause embryo loss or neonatal death. Therefore, rapid detection of the carriers is essential to manage these genetic defects. This study was conducted to develop a single-tube multiplex fluorescent amplification-refractory mutation system (mf-ARMS) PCR method for efficient genotyping of these 10 genetic defects and to compare its efficiency with the kompetitive allele specific PCR (KASP) genotyping assay. The mf-ARMS PCR method introduced 10 sets of tri-primers optimized with additional mismatches in the 3' end of wild and mutant-specific primers, size differentiation between wild and mutant-specific primers, fluorescent labeling of universal primers, adjustment of annealing temperatures and optimization of primer concentrations. The genotyping of 484 Holstein cows resulted in 16.12% carriers with at least one genetic defect, while no homozygous recessive genotype was detected. This study found carrier frequencies ranging from 0.0% (HH6) to 3.72% (HH3) for individual defects. The mf-ARMS PCR method demonstrated improved detection, time and cost efficiency compared with the KASP method for these defects. Therefore, the application of mf-ARMS PCR for genotyping Holstein cattle is anticipated to decrease the frequency of lethal alleles and limit the transmission of these genetic defects.

Sulfosuccinimidyl oleate ameliorates the high-fat diet-induced obesity syndrome by reducing intestinal and hepatic absorption.

Background: A high-fat Western diet is a risk factor for obesity and steatosis. Reducing intestinal absorption of a high-fat diet (HFD) is a feasible strategy to control obesity. Sulfosuccinimidyl oleate (SSO) inhibits intestinal fatty acid transport. Therefore, the aim of this study was to investigate the effects of SSO on HFD-induced glucose and lipid metabolism in mice and its possible underlying mechanisms. Methods: Male C57/BL were fed a HFD (60% calories) for 12 weeks and were administered an oral dose of SSO (50 mg/kg/day). The expression of lipid absorption genes (CD36, MTTP, and DGAT1) and the serum levels of triglycerides (TGs), total cholesterol (TC), and free fatty acids (FFAs) were detected. Lipid distribution in the liver was detected by oil red and hematoxylin and eosin staining. In addition, serum levels of inflammatory factors, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were measured to detect side effects. Results: SSO was effective in the treatment of obesity and metabolic syndrome induced by HFD in mice. It attenuated the assembly of intestinal epithelial chylomicrons by inhibiting intestinal epithelial transport and absorption of fatty acids, thereby reducing the gene expression levels of MTTP and DGAT1, resulting in decreased plasma TG and FFA levels. At the same time, it inhibited the transport of fatty acids in the liver and improved the steatosis induced by a HFD. The results of oil red staining showed that SSO treatment can reduce lipid accumulation in the liver by 70%, with no drug-induced liver injury detected on the basis of interleukin-6, C-reactive protein, ALT, and AST levels. In addition, SSO treatment significantly improved insulin resistance, decreased fasting blood glucose levels, and improved glucose tolerance in HFD-fed mice. Conclusion: SSO is effective in the treatment of obesity and metabolic syndrome induced by a HFD in mice. SSO reduces intestinal fatty acid absorption by reducing the inhibition of intestinal CD36 expression, followed by decreased TG and FFA levels, which attenuates HFD-induced fatty liver.

Which evaluation criteria of the short-term efficacy can better reflect the long-term outcomes for patients with nasopharyngeal carcinoma?

PURPOSE: To compare the consistency of one-dimensional Response Evaluation Criteria in Solid Tumors (1D-RECIST), two-dimensional WHO criteria (2D-WHO), and three-dimensional (3D) measurement for therapeutic response assessment of nasopharyngeal carcinoma (NPC). MATERIALS AND METHODS: Retrospective data of 288 newly diagnosed NPC patients were reviewed. Tumor size was assessed on magnetic resonance imaging (MRI) according to the 1D-RECIST, 2D-WHO, and 3D measurement criteria. Agreement between tumor responses was assessed using unweighted k statistics. The receiver operating characteristic (ROC) curve was used to determine the optimal cut-off point of the PTV. The Kaplan-Meier method and Cox regression were used for the survival analysis. RESULTS: The optimal cut-off point of the PTV for progression-free survival (PFS) was 29.6%. Agreement with PTV measurement was better for 1D measurement than for 2D and 3D measurements (kappa values of 0.646, 0.537, and 0.577 for 1D, 2D, and 3D measurements, respectively; P < 0.05). The area under the curve of the 1D measurement (AUC=0.596) was similar to that of the PTV measurement (AUC=0.621). Compared with 2D and 3D measurements, 1D measurement is superior for predicting prognosis in NPC (C-index of 0.672, 0.663, and 0.646 were for 1D, 2D, and 3D measurements, respectively; P < 0.005). Survival analysis showed that patients with non-responders had worse prognosis (P < 0.05). CONCLUSIONS: The 1D measurement more closely agreed with the PTV measurement than the 2D and 3D measurements for predicting therapeutic responses in NPC. Therefore, we recommend using the less time-consuming 1D-RECIST criteria in routine clinical practice.

Is it necessary for clinical tumor volume including neck muscles in target volume delineation of nasopharyngeal carcinoma?

OBJECTIVES: To calculate the shrinkage of the neck muscles and dosimetric changes and to clarify the necessity of covering part of the muscle in neck node region delineation for patients with nasopharyngeal carcinoma (NPC) treated with intensity-modulated radiotherapy. METHODS: In total, 44 patients with NPC were enrolled. Distances between the lateral border of the neck muscles and longitudinal midline were measured on every selected slice. This process was repeated three times, and the mean values of the three distances of planning computed tomography (CT) images and repeated CT images were adopted (labeled d1 and d2). The mean value of the differences between d1 and d2 was regarded as the medial shrinkage of the neck muscles. The initial clinical target volume of cervical lymph nodes (CTV-n) was shifted medially with the value of shrinkage, yielding a new CTV-n. Doses that covered 95% of the planning tumor volume (PTV) (D95), 99% of the PTV (D99), mean dose (Dmean), and maximum dose (Dmax) were used to calculate the dosimetric variation between the initial and new CTV-n. Comparisons were performed using the paired samples t test. RESULTS: The median d1 was 3.81 cm (range: 1.19-8.20 cm), and the median d2 was 3.68 cm (range: 0.94-9.59 cm), with a statistically significant difference (P < .001). The mean difference between d1 and d2 was 1.5 ± 3.1 mm (SD). The D95 and D99 of PTV of initial CTV-n decreased by 0.38% and 0.62% (P < .001 and P < .001, respectively). CONCLUSION: Patients with NPC experienced medial shrinkage of the neck muscles by 1.5 mm, and the consequent dose variation was negligible. It is unnecessary to cover part of the muscles in the delineation of the CTV-n.

Application of a treatment planning system-assisted large-aperture computed tomography simulator to percutaneous biopsy: initial experience of a radiation therapist.

OBJECTIVE: To evaluate the application of treatment planning system (TPS)-assisted large-aperture computed tomography (CT) simulator to percutaneous biopsy. METHODS: This retrospective study enrolled patients that underwent TPS-assisted large-aperture CT simulator-guided percutaneous biopsy from November 2018 to December 2019. Retrospective analyses of puncture accuracy were compared using paired t-test and a Wilcoxon rank sum test. The risk factors for puncture accuracy and complications were identified. RESULTS: A total of 38 patients were included in this study. There were no significant differences between the planned and actual puncture depth and angle. Pulmonary puncture was significantly associated with the accuracy of the puncture angle. The diagnostic rate of malignancy was 76% (29 of 38), of which 20 of 25 patients were in the group initially diagnosed with unconfirmed lesions and nine of 13 patients were in the group of treated patients that needed additional pathological analyses. For patients that underwent a pulmonary biopsy, 12 had minor pneumothorax and three suffered needle track bleeding. No other complications were observed. Regression analyses indicated a significant correlation between puncture angle and the incidence of pneumothorax. CONCLUSION: TPS-assisted large-aperture CT simulator may improve the percutaneous biopsy procedure by combining the advantages of radiotherapy specialties with computer targeting.

Outcomes and Experiences of Child-Bearing Women with Nasopharyngeal Carcinoma.

PURPOSE: Nasopharyngeal carcinoma (NPC) is more common among women in Southeast Asia. An important issue is whether it is safe for them to bear children after treatment and when it is safe to do so. We conducted this study to explore the relation between fertility and prognosis in child-bearing women with NPC. PATIENTS AND METHODS: Child-bearing women were defined as young women between the ages of 18 and 30. A total of 127 eligible child-bearing NPC patients were identified from December 2003 to December 2014. The patients were divided into two groups, depending on whether or not they had post-therapeutic births. The Kaplan-Meier method was used for survival analyses. The Log rank test was used to compare two survival curves and the independent significances of different prognostic factors were assessed by Cox proportional hazards regression analysis. RESULTS: The 5-year overall survival (OS) and disease-free survival (DFS) in the Childbirth group were significantly higher than those in the Non-Childbirth group (100% vs 88.8%, P = 0.026 and 100% vs 77.5%, P = 0.007, respectively). In the Childbirth group, no difference was found in the 5-year DFS between different birth interval times, from 1 to 5 years after treatment. The clinical stage was identified as the risk factor of OS (HR = 101.725, 95% CI: 2.160-4790.910, P = 0.019), and consequent childbirth after treatment was associated with favorable DFS (HR = 0.148, 95% CI: 0.034-0.643, P = 0.011). CONCLUSION: Post-therapeutic birth did not increase the mortality risk of child-bearing women with NPC. There was no significant correlation between the subsequent birth time window after treatment and the prognosis.

Effects of oral maintenance chemotherapy and predictive value of circulating EBV DNA in metastatic nasopharyngeal carcinoma.

BACKGROUND/OBJECTIVES: Oral maintenance chemotherapy can effectively prolong overall survival (OS) in many types of metastatic cancer, but its role in metastatic nasopharyngeal carcinoma (mNPC) is unclear. In this study, the efficacy of oral maintenance chemotherapy in mNPC and the effectiveness of circulating tumor EBV-DNA for screening patients were evaluated. METHODS: Between June 2016 and December 2017, 141 patients with mNPC who received platinum-based systemic chemotherapy were included (median follow-up time, 21 months). Patients were classified into two groups according to the administration of oral maintenance chemotherapy. Plasma samples were collected before, during, and after treatment for the measurement of circulating EBV DNA. RESULTS: The 2-year OS was higher for patients who received maintenance chemotherapy than for patients without maintenance chemotherapy (78.9% vs 62.7%, P = .016). Patients with undetectable posttreatment EBV-DNA after 4-6 cycles of systemic chemotherapy (n = 73) had a higher 2-year OS than that of patients with detectable EBV-DNA (n = 68) (82.16% vs 51.45%, P = .001). For patients with undetectable posttreatment EBV-DNA, OS was better for those with maintenance chemotherapy than for those without (86.7% vs 73%, P = .027). For patients with detectable posttreatment EBV-DNA, maintenance chemotherapy did not improve outcomes (49.5% vs 55.4%, P = .824). The most common acute events were hematological toxicity, and all were tolerable and curable. CONCLUSIONS: Oral maintenance chemotherapy with S1 or capecitabine can improve OS in mNPC. Posttreatment EBV-DNA was not only an independent prognosis factor for mNPC but also can screen out beneficiaries of maintenance chemotherapy.

Increased Duodenal Eosinophil Degranulation in Patients with Functional Dyspepsia: A Prospective Study.

Functional dyspepsia (FD) is a functional gastrointestinal disorder diagnosed by symptom-based criteria. It has been said that duodenal immune activation plays a role in the pathogenesis of FD. The primary aims of the study were to compare the total number of duodenal eosinophil and evaluate the eosinophil degranulation rate, number of duodenal degranulated eosinophil and mast cell between patients with FD and healthy subjects. We enrolled 96 patients with FD and 24 healthy controls at Sir Run Run Shaw Hospital. The total number of eosinophil was comparable in the second portion of duodenum (D2) and duodenal bulb (D1) between patients with FD and healthy controls (all P > 0.05). Significant higher eosinophil degranulation positive rate in D2 (P = 0.003) and a trend towards higher in D1 (P = 0.084) were observed in patients with FD compared with healthy controls. Moreover, the number of duodenal degranulated eosinophil in patients with FD were significantly increased than healthy controls in D1(9.8 ± 6.3 vs 2.9 ± 2.1 per HPF, P = 0.0002) and a trend towards increase in D2 (10.7 ± 7.7 vs 5.3 ± 0.9 per HPF, P = 0.077), respectively. However, degranulated mast cells in patients with FD were almost same with healthy controls. Increased eosinophils degranulation in duodenum play an important role in pathogenesis of FD.

[Nitric oxide mediated TNF-α, IL-1ß gene expression in liver induced by crush injury of rat's soft tissues].

OBJECTIVE: To explore the effect of nitric oxide (NO) on the gene expression of hepatic TNF-α and IL-1ß by crush injury of rat's soft tissues. METHODS: Rats were randomly divided into sham group, crush group, crush+aminoguanidine (AG) group, and crush+L-arginine (L-Arg) group. Activities of ALT and AST as well as NO level in serum were measured. Gene expressions of TNF-α and IL-1ß were detected with RT-PCR. RESULTS: Obvious increase in TNF-α and IL-1ß mRNA expression was detected in the crush group compared with the sham group (P<0.05). After pretreated L-Arg, expressions of TNF-α and IL-1ß mRNA were markedly increased (P<0.05). After pretreated AG, those indices obviously decreased (P<0.05). Activities of ALT and AST enhanced and NO level increased in the crush group compared with the sham group (P<0.05). Pretreatment with L-Arg or AG led to substantial increased or reduced activities of ALT and AST as well as NO levels, respectively. CONCLUSION: Endogenous NO mediated TNF-α, IL-1ß mRNA up expression in liver induced by increased production of NO after crush injury of rat's soft tissues.

Evaluation of specific marker CK13 and CK10/13 combined with APM staining for the diagnosis of amniotic fluid embolism and aspiration.

OBJECTIVE: To explore the value of CK13 (Rab) and CK10/13(Mab) as objective and specific biomarkers combined with Alcian-Phloxine-Martius yellow (APM) staining for the diagnosis of amniotic fluid embolism (AFE) and amniotic fluid aspiration (AFA). METHODS: A retrospective study of forensic autopsy cases of 148 neonatal deaths and 19 maternal deaths in the perinatal stage was conducted at the Institute of Pathology and Forensic Medicine, Zhejiang University. Medical records were reviewed and monoclonal antibody for CK13 (Rab) and CK10/13 (Mab) as immunostaining of amniotic fluid squamous cells, APM staining, and Hematoxylin and Eosin (HE) staining were used to diagnose the AFE and AFA. Descriptive statistics of the patient population were analyzed using SPSS 20.0 software. RESULTS: Immunoreactivity of CK13 and CK10/13 specifically identified squamous cells of all the AFE and AFA cases. The amniotic fluid squamous cells were stained positive in a deep brown color with the monoclonal antibody to CK 13 and CK10/13 whereas the endothelial cells and alveolar epithelial cells were negative. There was no CK13 or CK10/13 expression in the non-AFE and non-AFA cases. With APM staining keratinized squamous cells were pink and mucus was blue-green in marked contrast with the surrounding tissue, which improved the detection rates of both keratinized squamous cells and mucus. CONCLUSIONS: CK13 (Rab) and CK10/13 (Mab) are valuable and reliable biomarkers of amniotic fluid squamous cells. APM reveals enriched mucus and keratinized squamous cells of amniotic fluid. Immunohistochemical detection of CK13 and CK10/13 combined with APM staining can improve the accuracy and reduce the difficulty in the diagnosis of AFE and AFA.

The correlation between the comprehensive nutrition index and quality of life of patients with nasopharyngeal carcinoma treated by intensity-modulated radiotherapy.

The purpose of this study was to compare the changing tendency of nutrition with 54 nasopharyngeal carcinoma patients during intensity-modulated radiation therapy (IMRT), and to investigate the correlation between comprehensive nutritional status and quality of life (QoL), which was assessed by the European Organization for Research and Treatment of Cancer Core Quality-of-Life Questionnaire. The nutritional index, including body mass index, ideal body weight percentage, usual body weight percentage, albumin, hemoglobin, and total lymphocyte count (TLC), was evaluated at 2 time points: within 48 h after admission (T1) and at the end of treatment with IMRT (T2). A statistically significant downgrade of every index was observed during IMRT. A comprehensive nutritional model was established by principal components analysis at T2. QoL scores of functional (P = 0.002) and the global QoL scales (P = 0.001) existed a positive correlation with comprehensive nutritional status. QoL scores of symptom scales (P = 0.002) and 6 single items (P = 0.005) had a negative correlation with it. The scores of global QoL scales in comprehensive nutrition of normal (20.4%), moderate (55.6%), and severe malnutrition (24.1%) were 69.70 ± 17.98, 48.33 ± 19.25, and 37.18 ± 24.67, respectively. Patients with different nutritional status had different QoL (B = 10.405, SE = 2.828, t = 3.680, P = 0.001). Multiaspect nutritional supports should be enhanced to improve patients' comprehensive nutritional status during treatment.

[DNA extraction from bones and teeth using AutoMate Express forensic DNA extraction system].

OBJECTIVE: To explore a new method in order to extract DNA from bones and teeth automatically. METHODS: Samples of 33 bones and 15 teeth were acquired by freeze-mill method and manual method, respectively. DNA materials were extracted and quantified from the triturated samples by AutoMate Express forensic DNA extraction system. RESULTS: DNA extraction from bones and teeth were completed in 3 hours using the AutoMate Express forensic DNA extraction system. There was no statistical difference between the two methods in the DNA concentration of bones. Both bones and teeth got the good STR typing by freeze-mill method, and the DNA concentration of teeth was higher than those by manual method. CONCLUSION: AutoMate Express forensic DNA extraction system is a new method to extract DNA from bones and teeth, which can be applied in forensic practice.

[Risk factors of contrast-induced nephropathy after percutaneous coronary intervention in patients with coronary heart disease and the countermeasures].

OBJECTIVE: To identify the risk factors of contrast-induced nephropathy (CIN) after coronary interventional therapy (PCI) in patients with coronary heart disease and analyze the clinical outcomes and the preventive measures of CIN. METHODS: Ninety-one patients who developed CIN after PCI were retrospectively analyzed to identify the risk factors and explore the preventive measures. RESULTS: CIN was strongly associated with pre-procedural chronic renal failure, diabetes mellitus and administration of large-dose contrast. The incidence of cardiac mortality in hospital or in the follow-up at one year after PCI, and the incidence of myocardial infarction or major adverse cardiac events in the follow-up at one year were obviously higher in patients with CIN than those without CIN. CONCLUSION: Chronic renal failure, diabetes mellitus and large-dose contrast administration are 3 independent risk factors of CIN, which affects the prognosis of the patients. Reinforcement of a comprehensive perioperative management of PCI, especially a rigorous preoperative preparation, can be an important strategy for prevention of CIN.

Increased calcium deposits and decreased Ca2+ -ATPase in erythrocytes of ascitic broiler chickens.

The decrease of erythrocyte deformability may be one of the predisposing factors for pulmonary hypertension and ascites in broiler chickens. In mammals, the cytoplasmic calcium is a major regulator of erythrocyte deformability. In this study, the erythrocyte deformability was measured, and the precise locations of Ca2+ and Ca2+ -ATPase in the erythrocytes were investigated in chickens with ascites syndrome induced by low ambient temperature. The results showed that ascitic broilers had higher filtration index of erythrocyte compared with control groups, indicating a decrease in erythrocyte deformability in ascitic broilers. The more calcium deposits were observed in the erythrocytes of ascitic broilers compared with those of the age-matched control birds. The Ca2+ -ATPase reactive grains were significantly decreased on the erythrocyte membranes of ascitic broilers. Our data suggest that accumulation of intracellular calcium and inhibition of Ca2+ -ATPase might be important factors for the reduced deformability of the erythrocytes of ascitic broilers.

Validation of VKORC1 and CYP2C9 genotypes on interindividual warfarin maintenance dose: a prospective study in Chinese patients.

OBJECTIVES: To develop a warfarin-dosing algorithm that could be combined with pharmacogenomic and demographic factors, and to evaluate its effectiveness in a randomized prospective controlled clinical trial. METHODS: A pharmacogenetics-based dosing model was derived using retrospective data from 266 Chinese patients and multiple linear regression analysis. To prospectively validate this model, 156 patients with an operation of heart valve replacement were enrolled and randomly assigned to the group of pharmacogenetics-guided or traditional dosing for warfarin therapy. All patients were followed up for 50 days after initiation of warfarin therapy. The log-rank test was compared with the time-to-event (Kaplan-Meier) curves. Cox proportional hazards-regression model was used to assess the hazard ratio of the time to reach stable dose. RESULTS: The linear regression model derived from the pharmacogenomic model correlated with 54.1% of warfarin dosing variance. The final multiple linear regression model included age, body surface area, VKORC1, and CYP2C9 genotype. The study showed that the hazard ratio for the time to reach stable dose was 1.932 for the traditional dosing group versus the model-based group and a close and highly significant relationship was observed to exist between the predicted and the actual warfarin dose (R=0.454). CONCLUSION: A pharmacogenetics-based dosing algorithm has been developed for improvement in the time to reach the stable dosing of warfarin. This model may be useful in helping the clinicians to prescribe warfarin with greater safety and efficiency.

[Levels of C-reactive protein and antibodies against oxidized low-density lipoprotein in patients with different types of coronary heart disease].

OBJECTIVE: To investigate the relation of the levels of C-reactive protein (CRP) and antibodies against oxidized low-density lipoprotein (anti-oxLDL) to with acute coronary syndrome (ACS). METHODS: The levels of CRP, anti-oxLDL and anti-LDL were measured and compared in 96 subjects including 26 with acute myocardial infarction (AMI), 29 with unstable angina pectoris (UAP), 20 with stable angina pectoris (SAP) and 21 control subjects to evaluate the relationship between CRP and anti-oxLDL. RESULTS: Both CRP and anti-oxLDL levels in patients with ACS, including AMI and UAP, were significantly higher than those in SAP patients and the control subjects (P<0.05), but the level of anti-LDL showed no significant difference between the groups (P>0.05). There was significant positive correlation between the levels of CRP and anti-oxLDL (P<0.001). CONCLUSION: The specific immune response to oxLDL may play an important role in the instability of plaque and the occurrence of ACS, and anti-oxLDL level may serve as an important specific marker for the instability of plaque.