Application of improved GalNAc conjugation in development of cost-effective siRNA therapies targeting cardiovascular diseases.

N-Acetylgalactosamine (GalNAc)-conjugated small interfering RNA (siRNA) therapies have received approval for treating both orphan and prevalent diseases. To improve in vivo efficacy and streamline the chemical synthesis process for efficient and cost-effective manufacturing, we conducted this study to identify better designs of GalNAc-siRNA conjugates for therapeutic development. Here, we present data on redesigned GalNAc-based ligands conjugated with siRNAs against angiopoietin-like 3 (ANGPTL3) and lipoprotein (a) (Lp(a)), two target molecules with the potential to address large unmet medical needs in atherosclerotic cardiovascular diseases. By attaching a novel pyran-derived scaffold to serial monovalent GalNAc units before solid-phase oligonucleotide synthesis, we achieved increased GalNAc-siRNA production efficiency with fewer synthesis steps compared to the standard triantennary GalNAc construct L96. The improved GalNAc-siRNA conjugates demonstrated equivalent or superior in vivo efficacy compared to triantennary GalNAc-conjugated siRNAs.

Quality of childcare and delayed child development in left-behind children in China.

BACKGROUND: Inequalities in job opportunities and income prompts many Chinese parents to leave rural regions to work in urban regions. Their children are left behind in rural regions, subjected to worse quality of childcare that jeopardizes their development. This study aimed to examine the association between quality of childcare and delayed child development in under-three years children left behind in China. METHODS: Cross-sectional national survey was conducted in children left behind in rural China in 2017. Exploratory and confirmatory factor analysis was used to develop a quality of childcare index. Mutlilevel analyses determined factors associated with quality of childcare and child development on a province and individual level. RESULT: The largest population of at-risk children left behind were found in higher-GDP provinces. Children left behind had the lowest mean quality of childcare score. Multilevel analysis found that province level accounted for a great proportion of variance observed. CONCLUSIONS: While migration to urban regions for work may improve household income, a trade-off in worse quality of childcare and developmental delays exists. With improving household income often being the greatest contributing factor for parental migration, policies to reduce inequalities in job opportunities and wealth between rural and urban regions are required. IMPACT: Previous studies identified higher prevalence of developmental delays in children left behind in China. However, quality of childcare has not been examined. Based on WHO's Nurturing Care Framework, we developed a quality of childcare index to assess its association with child development in children left behind. Greatest proportion of children left behind at-risk of developmental delays resided in higher-GDP states, indicating a trade-off in worse quality of childcare and developmental delays. Since improving household income is the main factor for parental migration, policies to close inequalities in job opportunities and wealth between rural and urban regions are required.

Effect of Etomidate vs Propofol for Total Intravenous Anesthesia on Major Postoperative Complications in Older Patients: A Randomized Clinical Trial.

Importance: Older patients may benefit from the hemodynamic stability of etomidate for general anesthesia. However, it remains uncertain whether the potential for adrenocortical suppression with etomidate may increase morbidity. Objective: To test the primary hypothesis that etomidate vs propofol for anesthesia does not increase in-hospital morbidity after abdominal surgery in older patients. Design, Setting, and Participants: This multicenter, parallel-group, noninferiority randomized clinical trial (Etomidate vs Propofol for In-hospital Complications [EPIC]) was conducted between August 15, 2017, and November 20, 2020, at 22 tertiary hospitals in China. Participants were aged 65 to 80 years and were scheduled for elective abdominal surgery. Patients and outcome assessors were blinded to group allocation. Data analysis followed a modified intention-to-treat principle. Interventions: Patients were randomized 1:1 to receive either etomidate or propofol for general anesthesia by target-controlled infusion. Main Outcomes and Measures: Primary outcome was a composite of major in-hospital postoperative complications (with a noninferiority margin of 3%). Secondary outcomes included intraoperative hemodynamic measurements; postoperative adrenocortical hormone levels; self-reported postoperative pain, nausea, and vomiting; and mortality at postoperative months 6 and 12. Results: A total of 1944 participants were randomized, of whom 1917 (98.6%) completed the trial. Patients were randomized to the etomidate group (n = 967; mean [SD] age, 70.3 [4.0] years; 578 men [59.8%]) or propofol group (n = 950; mean [SD] age, 70.6 [4.2] years; 533 men [56.1%]). The primary end point occurred in 90 of 967 patients (9.3%) in the etomidate group and 83 of 950 patients (8.7%) in the propofol group, which met the noninferiority criterion (risk difference [RD], 0.6%; 95% CI, -1.6% to 2.7%; P = .66). In the etomidate group, mean (SD) cortisol levels were lower at the end of surgery (4.8 [2.7] µg/dL vs 6.1 [3.4] µg/dL; P < .001), and mean (SD) aldosterone levels were lower at the end of surgery (0.13 [0.05] ng/dL vs 0.15 [0.07] ng/dL; P = .02) and on postoperative day 1 (0.14 [0.04] ng/dL vs 0.16 [0.06] ng/dL; P = .001) compared with the propofol group. No difference in mortality was observed between the etomidate and propofol groups at postoperative month 6 (2.2% vs 3.0%; RD, -0.8%; 95% CI, -2.2% to 0.7%) and 12 (3.3% vs 3.9%; RD, -0.6%; 95% CI, -2.3% to 1.0%). More patients had pneumonia in the etomidate group than in the propofol group (2.0% vs 0.3%; RD, 1.7%; 95% CI, 0.7% to 2.8%; P = .001). Results were consistent in the per-protocol population. Conclusions and Relevance: Results of this trial showed that, compared with propofol, etomidate anesthesia did not increase overall major in-hospital morbidity after abdominal surgery in older patients, although it induced transient adrenocortical suppression. Trial Registration: ClinicalTrials.gov Identifier: NCT02910206.

miR-3064-5p and miR-4745-5p affect heparin sensitivity in patients undergoing cardiac surgery by regulating AT-III and factor X mRNA levels.

Subject: Perioperative regulation of coagulation function through heparin in patients undergoing cardiac surgery with cardiopulmonary bypass is an important part of performing cardiac surgery, and postoperative bleeding due to abnormal coagulation function caused by differences in heparin sensitivity in different individuals is an independent risk factor for postoperative complications and death. Method: Using an online database, 10 miRNAs interacting with AT-III and FX genes were predicted. Patients were divided into three groups according to the difference in activated clotting time (ACT) after the first dose of heparin (2.5 mg kg-1): group A: hyposensitive group (ACT < 480 s); group B: sensitive group (480 s ≤ ACT ≤ 760 s); and group C: hypersensitive group (ACT > 760 s). Perioperative and 24 h postoperative blood loss and other clinical data of patients in the three groups were recorded. Blood samples were collected before surgery, and RT-PCR was used to detect the levels of AT-III and FX gene mRNA and the levels of predicted 10 miRNAs. Result: Heparin sensitivity was positively correlated with AT-III mRNA levels and negatively correlated with FX gene mRNA levels in the three groups, and the blood loss in group B was significantly lower than that in groups A and C, which was statistically significant (p < 0.05). miR-3064-5p and miR-4745-5p expression levels were significantly different among group A, group B, and group C (p < 0.05) and were closely correlated with AT-III and FX gene mRNA expression levels, respectively. Conclusion: Differences in heparin sensitivity in patients undergoing cardiac surgery were associated with the mRNA expression of AT-III and FX genes, and the expression levels of miR-3064-5p and miR-4745-5p were found to be closely related to the AT-III and FX gene mRNA, respectively, indicating that miR-3064-5p and miR-4745-5p affect the differences in heparin sensitivity among different individuals by regulating the mRNA expression levels of AT-III and FX genes. Clinical Trial Registration: http://www.chictr.org.cn/abouten.aspx, identifier registration number: ChiCTR-2100047348.

Evaluation of a novel Cardiac Peri-Operative Transfusion Trigger Scoring system in patients with coronary artery disease.

BACKGROUND: A simple and accurate scoring system to guide perioperative blood transfusion in patients with coronary artery disease (CAD) undergoing cardiac surgery is lacking. The trigger point for blood transfusions for these patients may be different from existing transfusion guidelines. This study aimed to evaluate the safety and efficacy of a new scoring strategy for use in guiding transfusion decisions in patients with CAD. METHODS: A multicenter randomized controlled trial was conducted at three third-level grade-A hospitals from January 2015 to May 2018. Data of 254 patients in a Cardiac Peri-Operative Transfusion Trigger Score (cPOTTS) group and 246 patients in a group receiving conventional evaluation of the need for transfusion (conventional group) were analysed. The requirements for transfusion and the per capita consumption of red blood cells (RBCs) were compared between groups. RESULTS: Baseline characteristics of the two groups were comparable. Logistic regression analyses revealed no significant differences between the two groups in primary outcomes (1-year mortality and perioperative ischemic cardiac events), secondary outcomes (shock, infections, and renal impairment), ICU admission, and ICU stay duration. However, patients in the cPOTTS group had significantly shorter hospital stays, lower hospital costs, lower utilization rate and lower per capita consumption of transfused RBCs than controls. Stratified analyses revealed no significant differences between groups in associations between baseline characteristics and perioperative ischemic cardiac events, except for hemofiltration or dialysis and NYHA class in I. CONCLUSIONS: This novel scoring system offered a practical and straightforward guideline of perioperative blood transfusion in patients with CAD. Trial registration chiCTR1800016561(2017/7/19).

Heparin sensitivity and postoperative blood loss in patients undergoing cardiac surgery with cardiopulmonary bypass.

BACKGROUND: Heparin-associated coagulation disorder is an important factor related to postoperative bleeding in patients undergoing cardiac surgery with cardiopulmonary bypass. Currently, the relationship between heparin sensitivity and postoperative bleeding is unknown. OBJECTIVE: To investigate the relationship between individual heparin sensitivity and postoperative blood loss in patients undergoing cardiac surgery. DESIGN: Prospective controlled study. SETTING: Tertiary teaching hospital, Urumqi, Xinjiang, PR China. The study was conducted from January 2016 to August 2018. PATIENTS: A total of 195 adult patients undergoing cardiac valve replacement surgery were included. INTERVENTION: After initial heparin dosing (2.5 mg kg), patients were divided into three groups according to the whole blood activated clotting time (ACT): group A, insensitive group (ACT < 480 s); group B, sensitive group (480 s < ACT < 750 s); group C, hypersensitive group (ACT > 750 s). MAIN OUTCOME MEASURES: First, intra-operative and 24-h postoperative blood loss. Second, antithrombin (AT) and factor X mRNA levels. Third, the plasma levels of AT-III and factor X. Fourth, heparin sensitivity index. RESULTS: Blood loss was approximately 20 to 25% lower in group B than in groups A and C, which was statistically significant (P < 0.01). The AT-III mRNA levels increased from groups A to C and was positively associated with heparin sensitivity; the factor X mRNA levels changed in the opposite direction; a significant difference was observed between groups A and C (P < 0.05). The factor X plasma level showed the same trend as its mRNA. The AT-III plasma level was significantly lower in group B than in groups A and C (P < 0.05). CONCLUSION: Postoperative blood loss is related to heparin sensitivity in patients undergoing cardiac surgery, and the moderately sensitive patients have the least postoperative bleeding. Individual variation in heparin sensitivity is related to the mRNA and plasma levels of AT-III and factor X. TRIAL REGISTRATION: Registration number ChiCTR-RPC-17012259.

The key role of microtubules in hypoxia preconditioning-induced nuclear translocation of HIF-1α in rat cardiomyocytes.

BACKGROUND: Hypoxia-inducible factor (HIF)-1 is involved in the regulation of hypoxic preconditioning in cardiomyocytes. Under hypoxic conditions, HIF-1α accumulates and is translocated to the nucleus, where it forms an active complex with HIF-1ß and activates transcription of approximately 60 kinds of hypoxia-adaptive genes. Microtubules are hollow tubular structures in the cell that maintain cellular morphology and that transport substances. This study attempted to clarify the role of microtubule structure in the endonuclear aggregation of HIF-1α following hypoxic preconditioning of cardiomyocytes. METHODS: Primary rat cardiomyocytes were isolated and cultured. The cardiomyocyte culture system was used to establish a hypoxia model and a hypoxic preconditioning model. Interventions were performed on primary cardiomyocytes using a microtubule-depolymerizing agent and different concentrations of a microtubule stabilizer. The microtubule structure and the degree of HIF-1α nuclear aggregation were observed by confocal laser scanning microscopy. The expression of HIF-1α in the cytoplasm and nucleus was detected using Western blotting. Cardiomyocyte energy content, reflected by adenosine triphosphate/adenosine diphosphate (ATP/ADP), and key glycolytic enzymes were monitored by colorimetry and high-performance liquid chromatography (HPLC). Reactive oxygen species (ROS) production was also used to comprehensively assess whether microtubule stabilization can enhance the myocardial protective effect of hypoxic preconditioning. RESULTS: During prolonged hypoxia, it was found that the destruction of the microtubule network structure of cardiomyocytes was gradually aggravated. After this preconditioning, an abundance of HIF-1α was clustered in the nucleus. When the microtubules were depolymerized and hypoxia pretreatment was performed, HIF-1α clustering occurred around the nucleus, and HIF-1α nuclear expression was low. The levels of key glycolytic enzymes were significantly higher in the microtubule stabilizer group than in the hypoxia group. Additionally, the levels of lactate dehydrogenase and ROS were significantly lower in the microtubule stabilizer group than in the hypoxia group. CONCLUSION: The microtubules of cardiomyocytes may be involved in the process of HIF-1α endonuclear aggregation, helping to enhance the anti-hypoxic ability of cardiomyocytes.

The influence of rapamycin on the early cardioprotective effect of hypoxic preconditioning on cardiomyocytes.

INTRODUCTION: The purpose of this study was to examine the effects of rapamycin on the cardioprotective effect of hypoxic preconditioning (HPC) and on the mammalian target of rapamycin (mTOR)-mediated hypoxia-inducible factor 1 (HIF-1) signaling pathway. MATERIAL AND METHODS: Primary cardiomyocytes were isolated from rat pups and underwent rapamycin and/or HPC, followed by hypoxia/re-oxygenation (H/R) injury. Cell viability and cell injury were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays, and qRT-PCR was used to measure HIF-1α and mTOR mRNA expression. A Langendorff heart perfusion model was conducted to observe the effect of rapamycin. RESULTS: Rapamycin treatment nearly abolished the cardioprotective effect of HPC in cardiomyocytes, reduced cell viability (p = 0.007) and increased cell damage (p = 0.032). HIF-1α and mTOR mRNA expression increased in cardiomyocytes undergoing I/R injury within 2 h after HPC. After rapamycin treatment, mTOR mRNA expression and HPC-induced HIF-1α mRNA expression were both reduced (p < 0.001). A Langendorff heart perfusion model in rat hearts showed that rapamycin greatly attenuated the cardioprotective effect of HPC in terms of heart rate, LVDP, and dp/dtmax (all, p < 0.029). CONCLUSIONS: Rapamycin, through inhibition of mTOR, reduces the elevated HIF-1α expression at an early stage of HPC, and attenuates the early cardioprotective effect of HPC.

Prothrombotic SERPINC1 gene polymorphism may affect heparin sensitivity among different ethnicities of Chinese patients receiving heart surgery.

The purpose of this study was to investigate a possible correlation between single-nucleotide polymorphisms (SNPs) of the antithrombin (gene, SERPINC1, and perioperative sensitivity to heparin in patients receiving heart surgery. The SERPINC1 genotype and allele frequency, coagulation parameters 24 hours before and after surgery, and clinical findings were compared among 3 ethnic groups, Han, Uighur, and Kazakh, patientswho received heart surgery. In Han patients, longer coagulation time as well as higher heparin and protamine dosage was observed. SERPINC1 gene sequencing identified 2 mutations in exon 5, g.981A>G (rs5877) and g.1011A>G (rs5878). The minor allele frequency of allele (A>G) for rs5877 and rs5878 was higher in the Han patients and was significantly different among the ethnic groups (P = .004 and P = .006, respectively). The increased SERPINC1 SNP frequency among Han patients receiving heart surgery might contribute to the differences in their perioperative sensitivity to heparin.

Identification of a new SERPINC1 mutation in a Kazak family that alters the heparin binding capacity of antithrombin.

INTRODUCTION: Given its central role in mediating heparin-induced anti-coagulation, antithrombin (AT) gene mutations may result in heparin resistance. This study investigates the relationship between familial AT gene mutations and tolerance to heparin. METHODS: The medical history of a male patient with heparin resistance who received heart surgery and six of his family members was reviewed. Activated partial thromboplastin time (APTT), prothrombin time (PT), fibrinogen (Fib), D-dimer (D=D), and platelet count were determined to assess coagulation function. AT activity and the AT gene were also analyzed. For the newly identified gene mutations, polymorphisms were excluded in 120 healthy Kazak controls. RESULTS: Two mutations were identified in exon 7 of the AT gene, SERPINC1: g.1267G>A (p.A391T) found in five participants, including the index patient, and g.1334G>A, a silent mutation, in two family members. The g.1267G>A mutation may alter focal AT protein conformation. Neither of these mutations was observed in the healthy Kazak controls. Although all coagulation parameters and AT activity were within the normal ranges for the index patient and his family members, the platelet levels were significantly lower than that observed for the healthy Kazak controls (p=0.001). There was no significant difference in AT antigen levels between the groups; however, participants with the g.1267G>A mutation had a 44.25% reduction in heparin binding compared to the control group (p<0.001). CONCLUSION: We identified a novel hereditary mutation, g.1267G>A (p.A391T), in the AT gene, which reduces its heparin binding capacity and might be associated with resistance to heparin.

Prethrombotic state and cardiac events in patients with coronary heart disease during noncardiac surgery.

This study aimed to investigate the significance of the prethrombotic state (PTS) and 4 plasma markers in predicting perisurgical adverse cardiac events in patients with coronary heart disease (CHD) undergoing abdominal surgery. Perioperative adverse effects were recorded in 128 consecutive patients with CHD undergoing elective abdominal surgery. Plasma d-dimer, P-selectin, von Willebrand factor (VWF), and thrombus precursor protein were measured before and after the surgery. Patients with abnormal values in one or more of the 4 PTS markers were identified as having PTS, and data were analyzed by univariate and multivariate logistic regression. Abnormal presurgery levels of the 4 markers were found more frequently in those with adverse perioperative cardiac events than in those without. Multivariate analysis showed the odds ratios for adverse cardiac events to be 64.3 (PTS, P < .001), 25.7 (VWF, P = .003), and 23.5 (P-selectin, P = .04). Preoperative PTS is an independent risk factor for perioperative events in patients with CHD undergoing noncardiac surgery.

[Perioperative change in serum neutrophil gelatinase-associated lipocalin level among children with congenital heart disease and pulmonary hypertension].

OBJECTIVE: To study the perioperative change in serum neutrophil gelatinase-associated lipocalin (NGAL) level among children with congenital heart disease (CHD) and pulmonary hypertension (PH) and its significance. METHODS: Eighty children with CHD were divided into four groups according to pulmonary artery systolic pressure: non-PH, mild PH, moderate PH and severe PH groups. Serum NGAL levels were measured before operation, immediately after operation and 24 hours after operation. The relationship of serum NGAL level with PH and early prognosis was analyzed. RESULTS: The mild, moderate and severe PH groups had significantly higher serum NGAL levels than the non-PH group, and the severer the PH, the higher the serum NGAL level (P<0.01). All groups showed significant decreases in serum NGAL levels after operation (P<0.01). Serum NGAL level was positively correlated with the degree of PH and length of stay in the intensive care unit (P<0.01). CONCLUSIONS: Serum NGAL level increases in children with CHD and PH, and it gradually decreases after operation for closing the abnormal shunt. Serum NGAL level may be used as a serological indicator for evaluating the degree of PH and surgical outcome.

Goal-directed fluid therapy in gastrointestinal surgery in older coronary heart disease patients: randomized trial.

BACKGROUND: Our aim was to determine whether substitution of goal-directed fluid therapy (GDT) (perioperative fluid administration) for traditional therapy to manage elderly patients with coronary heart disease scheduled for gastrointestinal (GI) surgery was advantageous. We determined if it would reduce cardiac complications and shorten time to recovery and discharge. METHODS: Altogether, 60 of these elderly patients were randomized into GDT (n = 30) and control (n = 30) groups. In the GDT group, fluid management was carried out under guidance of hemodynamic status indicators. Types and quantities of fluids administered, blood loss, intraoperative urine output, time of extubation, intensive care unit (ICU) stay, hospital stay, postoperative adverse cardiac events, and GI complications were recorded. RESULTS: Total fluids infused were 2,910 ± 645 ml (GDT group) and 3,640 ± 771 ml (control group) (p < 0.05). Numbers of adverse cardiac events in the two groups were not significantly different (p = 0.121). Return of GI function was significantly faster in the GDT group (p < 0.001). Median ICU stay was 32.5 h in the GDT group and 47.5 h in the control group (p < 0.001). Median hospital stay was 18 days in the GDT group and 22 days in the control group (p < 0.001). CONCLUSIONS: GDT was associated with shorter ICU stay and time to discharge and faster return of GI function compared to traditional fluid therapy. The number of adverse cardiac events was similar in the two groups.

Preoperative HO-1 levels as prognostic factor for adverse cardiac events in elder patients undergoing non-cardiac surgery.

BACKGROUND: Hypoxia-inducible factor-1α (HIF-1α) and heme oxygenase-1(HO-1) are involved in the tissue hypoxic response. HYPOTHESIS: HIF-1α and HO-1 levels may predict cardiac ischemia and adverse cardiac events during non-cardiac surgery. METHODS: HIF-1α and HO-1 levels were determined in elderly patients undergoing non-cardiac surgery preoperatively and at 30 minutes, 48 and 72 hours postoperatively. Results were analyzed with respect to the occurrence of adverse cardiac events. RESULTS: A total of 380 patients with a mean age of 65.3 years were included, and 54 (14.2%) who had adverse cardiac events during or after the surgery. HIF-1α and HO-1 levels in the adverse cardiac event group were significantly higher than in the group without adverse cardiac events at each time point (all, P<0.05). In multivariates regression analysis, the odds of an adverse cardiac event was increased by every 1-year increase in age (odd ratio [OR] 1.39, P<0.001), abnormal ECG at baseline (OR 2.27, P = 0.048), myocardial infarction history (OR 3.18, P = 0.015), and positive baseline cTnI level were associated with an increased likelihood of an adverse cardiac event (OR 8.78, P = 0.019), and for every 1 unit increase of HO-1, the odds of an adverse cardiac event increased by 1.30 (P = 0.002). CONCLUSION: Determination of preoperative HO-1 levels may aid in identifying patients at risk of developing ischemic cardiac events.

[A meta-analysis on efficacy of antifibrinolytic agents during perioperative period in patients undergoing coronary artery bypass grafting treated with antiplatelet agents].

OBJECTIVE: To evaluate the efficacy of antifibrinolytic agents in coronary artery bypass grafting (CABG) patients receiving antiplatelet. METHODS: We searched PubMed, EMbase, Highwire, CENTREN and its affiliated clinical trial registration data center, CBMdisc and CNKI databases from 2000 to 2010. Randomized controlled trials investigating the efficacy of anti-fibrinolytic agents (aprotinin and tranexamic acid) in CABG patients were identified. Study selection and meta-analysis were conducted according to the Cochrane Handbook for systematic reviews. Date were extracted from these trials by 2 reviewers independently and analyzed by RevMan 5.0 software. RESULT: Eleven RCT trials (n = 725) were included and data confirmed the efficacy of antifibrinolytic therapy in terms of reducing bleeding within 24 hours after operation (MD = -306.5 ml, 95%CI: -351.52 to -261.52, P < 0.01), number of patients who need blood transfusion (OR = 0.37, 95%CI: 0.26 to 0.51, P < 0.01), amount of blood transfusion (MD = -0.59 U, 95%CI: -0.69 to -0.50, P < 0.01), surgical re-exploration(OR = 0.27, 95%CI: 0.09 to 0.78, P = 0.02), and thrombotic events (OR = 0.49, 95%CI: 0.25 to 0.97, P = 0.04) in CABG patients receiving antiplatelet, while compared with blank treatment. CONCLUSION: This analysis showed that antifibrinolytic agents are effective for reducing bleeding within 24 hours after operation, amount of blood transfusion, surgical re-exploration and do not increase the incidence of thrombotic events in CABG patients receiving antiplatelet before operation.

Identification of 12 animal species meat by T-RFLP on the 12S rRNA gene.

The verification of authenticity of meat products is relevant for economical, religious or public health concerning reasons. A molecular approach using terminal restriction fragment length polymorphism (T-RFLP) was developed to distinguish 12 common economically important meat species. The partial 12S rRNA gene was amplified with double-fluorescently labeled primers. The amplified fragments were digested with two endonucleases and only the terminal restriction fragment containing labeled primer was detected on capillary electrophoresis system ABI3100. AluI and Tru9I generated differently-sized terminal fragments in different species. Pig and buffalo can be separated by 3'-terminal fragment of AluI digestion. Horse, turkey, goat, sheep, deer, and cattle can be further separated by 5'-terminal fragment of Tru9I digestion. Dog and chicken, sturgeon and salmon can finally be separated by 5'-terminal fragment of AluI digestion and 3'-terminal fragment of Tru9I digestion. Our results demonstrated the potential feasibility and applicability of T-RFLP method for rapid and accurate identification of animal species.

[Effects of alligator Zhikegao on relieving cough dispelling phlegm and anti-inflammation].

OBJECTIVE: To research the effects of Alligator Zhikegao on relieving cough, dispelling phlegm and anti-inflammation. METHOD: The coughing tests in mice, the phenol red secreting tests in mice, ear edema tests in mice,and paw edema tests and subcutaneous cotton ball granuloma in rats were adopted for observing the related pharmacological effects of Alligator Zhikegao. RESULT: Alligator Zhikegao could obviously prolong the latent period and decrease the times of mouse coughing, and remarkably inhibit the mouse ear edema (P < 0.001), the rat paw edema and the hyperplasia of subcutaneous cotton ball granuloma in rats. Alligator Zhikegao 11.70 g x kg(-1) could significant improve the carbonic clearances of macrophages (P <0.05) and the hemolysin level in serum (P <0.01). CONCLUSION: Alligator Zhikegao has significant effects on relieving cough, dispelling phlegm, anti-inflammation and immunological regulation.

Anthocyanins increase low-density lipoprotein and plasma cholesterol and do not reduce atherosclerosis in Watanabe Heritable Hyperlipidemic rabbits.

Anthocyanin-rich beverages have shown beneficial effects on coronary heart disease in epidemiological and intervention studies. In the present study, we investigated the effect of black currant anthocyanins on atherosclerosis. Watanabe Heritable Hyperlipidemic rabbits (n = 61) were fed either a purified anthocyanin fraction from black currants, a black currant juice, probucol or control diet for 16 weeks. Purified anthocyanins significantly increased plasma cholesterol and low-density lipoprotein (LDL) cholesterol. Intake of black currant juice had no effect on total plasma cholesterol, but lowered very-low-density lipoprotein (VLDL) cholesterol significantly. There were no significant effects of either purified anthocyanins or black currant juice on aortic cholesterol or development of atherosclerosis after 16 weeks. Probucol had no effect on plasma cholesterol but significantly lowered VLDL-cholesterol and decreased aortic cholesterol accumulation. The erythrocyte antioxidant enzyme glutathione peroxidase was significantly increased by purified anthocyanins and superoxide dismutase was increased by both anthocyanin-containing treatments. Other markers of plasma antioxidant capacity, antioxidant enzymes, protein and lipid oxidation were not affected by any of the anthocyanin treatments. Adverse effects of purified anthocyanins were observed on plasma- and LDL-cholesterol. These effects were not observed with black currant juice, suggesting that black currants may contain components reducing the adverse effects of anthocyanins.

Fast and robust simultaneous determination of three veterinary antibiotics in groundwater and surface water using a tandem solid-phase extraction with high-performance liquid chromatography-UV detection.

A simple and robust analytical method is presented in which the three veterinary antibiotics oxytetracycline (OTC), sulfachloropyridazine (SCP) and tylosin (TYL) were simultaneously determined in surface water and groundwater. The three compounds were simultaneously extracted from the water samples using a mixture of methanol, EDTA and McIlvaine buffer (citric acid and sodium orthophosphate) and then cleaned-up and pre-concentrated by solid-phase extraction using sacrificial Isolute strong anion-exchange cartridges, to remove interfering organic material, and Waters Oasis hydrophilic-liphophilic balance polymer cartridges, to retain the compounds, in tandem. Analysis was performed using liquid chromatography with ultraviolet detection. Recoveries for river water samples spiked at 10 and 1 microgl(-1) were respectively 99.6+/-4.6 and 99.4+/-8.4% for OTC; 99.9+/-2.2 and 105.0+/-5.7% for SCP; and 94.9+/-2.4 and 71.6+/-8.2% for TYL. Overall limits of detection based on pre-concentrating 400 ml of sample were 0.35 microgl(-1) for OTC and TYL and 0.25 microgl(-1) for SCP.

Ultrasonic extraction of veterinary antibiotics from soils and pig slurry with SPE clean-up and LC-UV and fluorescence detection.

A simple and rapid analytical method is presented in which the three veterinary antibiotics oxytetracycline (OTC), sulfachloropyridazine (SCP) and tylosin (TYL) are simultaneously extracted and determined in four different soils. Extractions were carried out by a combination of ultrasonic agitation and vortex mixing using a mixture of methanol, EDTA and McIlvaine buffer at pH 7 as the extractant solution. The extracts were then cleaned-up by a tandem solid phase extraction (SPE) method using an Isolute SAX anion exchange cartridge to remove natural organic matter and an Oasis HLB polymeric cartridge to retain the study compounds. Analysis was by HPLC-UV with additional fluorescence detection for SCP. Recoveries were in the range 68-85% for SCP in all soil types, 58-75% for OTC in sandy soils, 27-51% for OTC in clay containing soils, 74-105% for TYL and 47-61% in a clay soil. OTC and SCP were also extracted from liquid pig manure using a mixture of EDTA and McIlvaine buffer at pH 7 with ultrasonic agitation and vortex mixing with SPE clean-up and HPLC-UV analysis. Recoveries were greater than 77% and 58% for OTC and SCP, respectively. Limits of detection were 18mugkg(-1) for OTC and SCP and 40mugkg(-1) for TYL in soils and 70mugL(-1) for OTC and 140mugL(-1) for SCP in pig slurry.