Use of dual priming oligonucleotide system-based multiplex RT-PCR combined with high performance liquid chromatography assay for simultaneous detection of five enteric viruses associated with acute enteritis.

In this study, a specific and sensitive method for simultaneous detection of human astrovirus, human rotavirus, norovirus, sapovirus and enteric adenovirus associated with acute enteritis was developed, based on the specific dual priming oligonucleotide (DPO) system and the sensitive high-performance liquid chromatography (HPLC) analysis. The DPO system-based multiplex reverse transcription-polymerase chain reaction (RT-PCR) combined with HPLC assay was more sensitive than agarose gel electrophoresis analysis and real-time SYBR Green PCR assay, and showed a specificity of 100% and sensitivity of 96%-100%. The high sensitivity and specificity of the assay indicates its great potential to be a useful tool for the accurate diagnosis of enteric virus infections.

Efficient L-Alanine Production by a Thermo-Regulated Switch in Escherichia coli.

L-Alanine has important applications in food, pharmaceutical and veterinary and is used as a substrate for production of engineered thermoplastics. Microbial fermentation could reduce the production cost and promote the application of L-alanine. However, the presence of L-alanine significantly inhibit cell growth rate and cause a decrease in the ultimate L-alanine productivity. For efficient L-alanine production, a thermo-regulated genetic switch was designed to dynamically control the expression of L-alanine dehydrogenase (alaD) from Geobacillus stearothermophilus on the Escherichia coli B0016-060BC chromosome. The optimal cultivation conditions for the genetically switched alanine production using B0016-060BC were the following: an aerobic growth phase at 33 °C with a 1-h thermo-induction at 42 °C followed by an oxygen-limited phase at 42 °C. In a bioreactor experiment using the scaled-up conditions optimized in a shake flask, B0016-060BC accumulated 50.3 g biomass/100 g glucose during the aerobic growth phase and 96 g alanine/100 g glucose during the oxygen-limited phase, respectively. The L-alanine titer reached 120.8 g/l with higher overall and oxygen-limited volumetric productivities of 3.09 and 4.18 g/l h, respectively, using glucose as the sole carbon source. Efficient cell growth and L-alanine production were reached separately, by switching cultivation temperature. The results revealed the application of a thermo-regulated strategy for heterologous metabolic production and pointed to strategies for improving L-alanine production.

Recombinant lactobacillus expressing G protein of spring viremia of carp virus (SVCV) combined with ORF81 protein of koi herpesvirus (KHV): A promising way to induce protective immunity against SVCV and KHV infection in cyprinid fish via oral vaccination.

Spring viremia of carp virus (SVCV) and koi herpesvirus (KHV) are highly contagious and pathogenic to cyprinid fish, causing enormous economic losses in aquaculture. Although DNA vaccines reported in recent years could induce protective immune responses in carps against these viruses via injection, there are a number of consequences and uncertainties related to DNA vaccination. Therefore, more effective and practical method to induce protective immunity such as oral administration would be highly desirable. In this study, we investigated the utilities of a genetically engineered Lactobacillus plantarum (L. plantarum) coexpressing glycoprotein (G) of SVCV and ORF81 protein of KHV as oral vaccine to induce protective immunity in carps via oral vaccination. The surface-displayed recombinant plasmid pYG-G-ORF81 was electroporated into L. plantarum, giving rise to LP/pYG-G-ORF81, where expression and localization of G-ORF81 fusion protein from the LP/pYG-G-ORF81 was identified by SDS-PAGE, Western blotting and immunofluorescence assay. Bait feed particles containing the LP/pYG-G-ORF81 were used as vaccine to immunize carps via gastrointestinal route. Compared to control groups, the carps orally immunized with the LP/pYG-G-ORF81 were induced significant levels of immunoglobulin M (IgM), and its immunogenicity was confirmed by viral loads reduction detected by PCR assay after virus challenge followed by an effective protection rate 71% in vaccinated carps and 53% in vaccinated koi until at days 65 post challenge, respectively. Our study here demonstrates, for the first time, the ability of recombinant L. plantarum as oral vaccine against SVCV and KHV infection in carps, suggesting a practical multivalent strategy for the control of spring viremia of carp and koi herpesvirus disease.

Immunogenicity in Swine of Orally Administered Recombinant Lactobacillus plantarum Expressing Classical Swine Fever Virus E2 Protein in Conjunction with Thymosin α-1 as an Adjuvant.

Classical swine fever, caused by classical swine fever virus (CSFV), is a highly contagious disease that results in enormous economic losses in pig industries. The E2 protein is one of the main structural proteins of CSFV and is capable of inducing CSFV-neutralizing antibodies and cytotoxic T lymphocyte (CTL) activities in vivo. Thymosin α-1 (Tα1), an immune-modifier peptide, plays a very important role in the cellular immune response. In this study, genetically engineered Lactobacillus plantarum bacteria expressing CSFV E2 protein alone (L. plantarum/pYG-E2) and in combination with Tα1 (L. plantarum/pYG-E2-Tα1) were developed, and the immunogenicity of each as an oral vaccine to induce protective immunity against CSFV in pigs was evaluated. The results showed that recombinant L. plantarum/pYG-E2 and L. plantarum/pYG-E2-Tα1 were both able to effectively induce protective immune responses in pigs against CSFV infection by eliciting immunoglobulin A (IgA)-based mucosal, immunoglobulin G (IgG)-based humoral, and CTL-based cellular immune responses via oral vaccination. Significant differences (P < 0.05) in the levels of immune responses were observed between L. plantarum/pYG-E2-Tα1 and L. plantarum/pYG-E2, suggesting a better immunogenicity of L. plantarum/pYG-E2-Tα1 as a result of the Tα1 molecular adjuvant that can enhance immune responsiveness and augment specific lymphocyte functions. Our data suggest that the recombinant Lactobacillus microecological agent expressing CSFV E2 protein combined with Tα1 as an adjuvant provides a promising strategy for vaccine development against CSFV.

[Changes of dietary pattern among adults in Liaoning province, 1989 to 2006].

OBJECTIVE: To study the changes of dietary pattern among adult residents in different areas of Liaoning province from 1989 to 2006. METHODS: Healthy adults (6213 subjects) at age of 18 - 65 years from 480 households in three cities (Shenyang, Yingkou, Wafangdian) and three counties (Qingyuan, Huanren, Chaoyang) were selected with stratified multiple cluster random sampling. The information on nutrient intake of the subjects were collected from datasets of Chinese Health and Nutrition Survey conducted in 1989, 1991, 1993, 2000, 2004, and 2006. Different food intake, the nutrients intake percentages for recommended nutrition intake (RNI) and appropriate intake (AI), and the percentages of total energy and protein from grain, animal product, bean and its product were calculated to assess the residents' dietary pattern and nutrition status. The changes of dietary pattern among adult residents were analyzed. RESULTS: Among the residents, there were a 38.1% of decreased intake for grain (from 601.9 to 372.5 g/d), 20.5% for potato (from 75.6 to 60.1 g/d), 25.1% for beans (from 38.7 to 29.0 g/d), and a 77.2% of increased intake for fish and shrimp (from 25.0 to 44.3 g/d), 36.9% for livestock and poultry (from 65.6 to 89.8 g/d), 47.7% for fruit (from 70.7 to 104.4 g/d), and intake of milk product (from 5.8 to 21.3 g/d), egg (from 17.3 to 35.7 g/d), vegetable (from 296.1 to 316.3 g/d) were also increased from 1989 to 2006. During the period, the intake percentages of energy and protein from grain decreased from 67.5% (8.7 MJ/12.8 MJ per day) to 51.5% (5.0 MJ/9.6 MJ per day) and from 72.0% (66.2 g/91.9 g per day) to 59.7% (45.3 g/75.9 g per day), and on the contrary those from animal products increased from 8.9% (1.1 MJ/12.8 MJ per day) to 14.8 (1.4 MJ/9.6 MJ per day) and from 15.9% (14.6 g/91.9 g per day) to 27.9% (21.2 g/75.9 g per day), respectively. In 2006, the intake of vitamin A (508.9 µg/d) was 67.6% of it's RNI, intake of vitamin B(2) (0.9 mg/d) was 64.6% and the intake of calcium (453.7 mg/d) was 52.5% of it's AI among the residents. CONCLUSION: The intake of plant food decreased and that of animal food increased from 1989 to 2006 and the dietary intakes of calcium, vitamin A, vitamin B(2) need to be increased among adult population of Liaoning province.

Initial experiences of maintaining atrioventricular intrinsic conduction during cardiac resynchronization therapy in non-responders.

BACKGROUND: Cardiac resynchronization therapy (CRT) is a major breakthrough in therapy for advanced heart failure patients; however, a number of key clinical research questions remain, perhaps most importantly the issue of why apparently suitable patients do not respond to CRT. METHODS: Seven patients, six males and one female, aged (56.43 +/- 6.13) years, all diagnosed with dilated cardiomyopathy, were included in this study. They were all non-responders to CRT who underwent routine optimization postoperatively, and received optimal drug therapy. On the basis of biventricular pacing, titrating various atrioventricular (AV) intervals were performed to get the true fusional QRS complexes composed of biventricular pacing and AV intrinsic conduction. Then, the effects of AV intrinsic conduction during CRT were evaluated. RESULTS: On the setting of AV intrinsic conduction during CRT, the true fusional QRS complexes were the narrowest, and all patients showed alleviation of symptoms, improvement of exercise tolerance, life quality and hemodynamic parameters during more than 6 months of follow-up. CONCLUSIONS: Titrating AV intervals to get the true fusional QRS complexes composed of biventricular pacing and AV intrinsic conduction will be beneficial for non-responders to CRT. Maintaining AV intrinsic conduction during CRT may decrease the rates of non-responders to CRT.

[The prospective study on the prognosis in patients with ventricular tachycardia or fibrillation treated with implantable automatic cardioverter defibrillator].

OBJECTIVE: To explore the effect of implantable automatic cardioverter defibrillator (ICD) on improvement of the prognosis of patients with ventricular tachycardia or fibrillation (VT/VF). To compare the advantages and disadvantages of ICD with antiarrhythmic drug, to select the best indication of ICD and review the protocol of the following-up of ICD patients, and present scientific evidence for the morebroad popularization who needs ICD in China. METHODS: In 99 selected patients who had the indication of class I, 27 patients were treated by ICD (ICD group), and 72 patients were not (non-ICD group). Patients in the two groups had the similar basic clinical characteristics. The incidence of syncope, CPR, and VF in ICD group were more common than those in non-ICD group. Patients in the two groups received same basic therapy. The total mortality rate and the incidence of cardiac events in two groups were compared in 3 months, 6 months, 12 months and 15 months. RESULTS: The total mortality rate and the incidence of cardiac events in ICD group were significantly lower than those in non-ICD group in the follow-up period. The mortality rate in ICD group is 0, and the mortality rate in non-ICD group is 20.8%. CONCLUSIONS: (1) ICD treatment can decrease the incidence of cardiac events of patients with high risk of sudden cardiac death (SCD), and improve their survival. (2) The key measures to insure the efficacy, safety, and cost-effectiveness of ICD treatment is: to select the patients correctly, to optimize the implanting process and the follow-up, and use rational assistant therapy.

[Prognostic value of microvolt level T-wave alternans in patients at high risk of sudden cardiac death].

OBJECTIVE: To explore the predict value of microvolt level T-wave alternans (MTWA) for malignant ventricular arrhythmia (MVA) and sudden cardiac death (SCD) in high risk patients. METHODS: A total of 105 healthy subjects (control group) and 138 patients with history of VT or VF or patients with LVEF < or = 45% (SCD high risk group) were included in this study (mean age 52 years old). MTWA, LVEF, HRV, NSVT, QRS, QTc values and MACE data (death, causes of death, MVA, re-hospitalization, syncope) during follow up (12.0 +/- 1.3) months were obtained. RESULTS: The normal reference value of MTWA was defined as < 37 microV. Positive rate in SCD high risk group was significantly higher than that in control group (45.7% vs. 4.8%, P < 0.01). There was no cardiovascular event report in control group. In SCD high risk group, there were 11 deaths (MTWA positive rate 81.8%) including 7 SCD (MTWA positive rate 85.7%), 17 MVA (MTWA positive rate 88.2%), 9 cases of syncope (MTWA positive rate 77.8%), 21 cases of re-hospitalization during the follow up (MTWA positive rate 85.7%). Logistic regression analysis revealed that positive MTWA, a history of myocardial infarction and LVDd > or = 60 mm were risk factors for all cause of death and positive MTWA was the only factor to predict SCD. The factors related to MVA in turn were positive MTWA, LVEF < or = 35%, a history of cardiopulmonary resuscitation and a history of syncope. Positive MTWA and LVEF < or = 35% are the independent risk factors for predicting MVA (P < 0.01). The sensitivity was 91% and specifity was 66% by combined use of positive MTWA and LVEF < or = 35% to predict MVA. MTWA positive rates were 68.3% and 87.5% respectively in 41 ICD patients and ICD patients with automatic shock during follow up. CONCLUSION: Non-invasive MTWA measurement could be used as a screening tool to predict SCD or MVA in high risk patients.

HC-Pro protein of sugar cane mosaic virus interacts specifically with maize ferredoxin-5 in vitro and in planta.

Symptom development of a plant viral disease is a result of molecular interactions between the virus and its host plant; thus, the elucidation of specific interactions is a prerequisite to reveal the mechanism of viral pathogenesis. Here, we show that the chloroplast precursor of ferredoxin-5 (Fd V) from maize (Zea mays) interacts with the multifunctional HC-Pro protein of sugar cane mosaic virus (SCMV) in yeast, Nicotiana benthamiana cells and maize protoplasts. Our results demonstrate that the transit peptide rather than the mature protein of Fd V precursor could interact with both N-terminal (residues 1-100) and C-terminal (residues 301-460) fragments, but not the middle part (residues 101-300), of HC-Pro. In addition, SCMV HC-Pro interacted only with Fd V, and not with the other two photosynthetic ferredoxin isoproteins (Fd I and Fd II) from maize plants. SCMV infection significantly downregulated the level of Fd V mRNA in maize plants; however, no obvious changes were observed in levels of Fd I and Fd II mRNA. These results suggest that SCMV HC-Pro interacts specifically with maize Fd V and that this interaction may disturb the post-translational import of Fd V into maize bundle-sheath cell chloroplasts, which could lead to the perturbation of chloroplast structure and function.

Body iron stores and dietary iron intake in relation to diabetes in adults in North China.

OBJECTIVE: To evaluate the association between body iron stores, dietary iron intake, and risk of diabetes in northern China. RESEARCH DESIGN AND METHODS: The data of a cross-sectional household survey in 2002 in Liaoning Province in northern China was used. The final sample in our study contained 2,997 subjects aged >or=18 years. Fasting plasma glucose and serum ferritin were measured. Dietary information was collected by 3-day food records. RESULTS: Serum ferritin was associated with elevated risk of diabetes even adjusted for age, sex, nondietary factors, and dietary factors. No association among total iron intake, nonheme iron intake, and diabetes risk was found. However, higher heme iron intake was significantly associated with elevated risk of diabetes after adjusting for known factors. CONCLUSIONS: In Chinese, associations among higher serum ferritin level, higher heme iron intake, and elevated risk of diabetes were found.

[Efficacy of implantable loop recorders in establishing symptom-rhythm correlation in patients with unexplained syncope].

OBJECTIVE: To evaluate the efficacy of the implantable loop recorder (ILR) in establishing symptom-rhythm correlation in patients with unexplained syncope. METHODS: Implantable loop recorders (ILR, Reveal Plus(9526), Medtronic Inc.) were implanted in 10 patients [aged 14 - 78 (41 +/- 22) years, 6 female] with unexplained syncope from October 2002 to May 2005. Syncopal episodes were (4.5 +/- 1.4) patients. RESULTS: During the monitoring period [8 - 21 (15.3 +/- 3.6) months], there were 24 times syncopal episodes in 6 patients. A total of 211 arrhythmia events were documented by ILR in 7 patients and symptom-rhythm correlation could be established in these 7 patients. In 2 patients, there were no recurrent syncopes and no arrhythmia events could be recorded. In 1 patient, syncope was caused by reasons other than arrhythmia. CONCLUSION: ILR is useful in determining the presence or absence of an arrhythmia during symptoms of syncope when conventional diagnostic testing, such as electrocardiogram, Holter monitoring, and/or external loop recording, is inconclusive.