Immunoelectron Microscopy for Locating Calvin Cycle Enzymes in the Thylakoids of Synechocystis 6803

Unicellular cyanobacteria Synechocystis 6803 were fixed using high-pressure freezing （HPF） and freeze substitution without any chemical cross-linkers. Immunoelectron microscopy of these cells showed that five sequential enzymes of the Calvin cycle （phosphoriboisomerase, phosphoribulokinase, ribulose-1,5-bisphosphate carboxylase/oxygenase （RuBisCO）, 3-phosphoglyceratekinase and glyceraldehyde-3-phosphate dehydrogenase） and the catalytic portion of the chloroplast H^＋-ATP synthase （CF1） are located adjacent to the thylakoid membranes. Cell-free extracts of Synechocystis were processed by ultracentrifugation to isolate thylakoid fractions sedimenting at 40 000, 90 000, and 150 000 g. Among these, the 150 000-g fraction showed the highest linked activity of the above five sequential Calvin cycle enzymes and also the highest coordinated activity of light and dark reactions as assessed by ribose-5-phosphate （R-5-P） ＋ADP dependent CO2 fixation. Immunogold labeling of this membrane fraction confirmed the presence of the above five enzymes as well as the catalytic portion of the CF1 ATP synthase. Notably, the protein A-gold labeling of the thylakoids was observed without use of chemical cross-linkers and in spite of the normal washing steps used during standard immunolabeling. The results showed that soluble Calvin cycle enzymes might be organized along the thylakoid membranes.     

Metabolomics and Incidence of Atrial Fibrillation in African Americans: The Atherosclerosis Risk in Communities (ARIC) Study

Background

Atrial fibrillation (AF) is a common arrhythmia. Application of metabolomic approaches, which may identify novel pathways and biomarkers of disease risk, to a longitudinal epidemiologic study of AF has been limited.

Methods

We determined the prospective association of 118 serum metabolites identified through untargeted metabolomics profiling with the incidence of newly-diagnosed AF in 1919 African-American men and women from the Atherosclerosis Risk in Communities study without AF at baseline (1987–1989). Incident AF cases through 2011 were ascertained from study electrocardiograms, hospital discharge codes, and death certificates.

Results

During a median follow-up of 22 years, we identified 183 incident AF cases. In Cox proportional hazards models adjusted for age, sex, smoking, body mass index, systolic blood pressure, use of antihypertensive medication, diabetes, prevalent heart failure, prevalent coronary heart disease, and kidney function, two conjugated bile acids (glycolithocholate sulfate and glycocholenate sulfate) were significantly associated with AF risk after correcting for multiple comparisons (p<0.0004). Multivariable-adjusted hazard ratios (95% confidence intervals) of AF were 1.22 (1.12–1.32) for glycolithocholate sulfate and 1.22 (1.10–1.35) for glycocholenate sulfate per 1-standard deviation higher levels. Associations were not appreciably different after additional adjustment for alcohol consumption or concentrations of circulating albumin and liver enzymes.

Conclusion

We found an association of higher levels of two bile acids with an increased risk of AF, pointing to a potential novel pathway in AF pathogenesis. Replication of results in independent studies is warranted.     

Diagnostic accuracy of the TIMI risk score in patients with chest pain in the emergency department: a meta-analysis

Background

The Thrombolysis in Myocardial Infarction (TIMI) risk score uses clinical data to predict the short-term risk of acute myocardial infarction, coronary revascularization or death from any cause. It was originally developed for use in patients with unstable angina or non–ST-elevation myocardial infarction. We sought to expand the clinical application of the TIMI risk score by assessing its prognostic accuracy in patients in the emergency department with potential acute coronary syndromes.

Methods

We searched five electronic databases, hand-searched reference lists of included studies and contacted content experts to identify articles for review. We included prospective cohort studies that validated the TIMI risk score in emergency department patients. We performed a meta-regression to determine whether a linear relation exists between TIMI risk score and the cumulative incidence of cardiac events.

Results

We included 10 prospective cohort studies (with a total of 17 265 patients) in our systematic review. Data were available for meta-analysis in 8 of the 10 studies. Of patients with a score of zero, 1.8% had a cardiac event within 30 days (sensitivity 97.2%, 95% CI 96.4–97.8; specificity 25.0%, 95% CI 24.3–25.7; positive likelihood ratio 1.30, 95% CI 1.28–1.31; negative likelihood ratio 0.11, 95% CI 0.09–0.15). Meta-regression analysis revealed a strong linear relation between TIMI risk score (p < 0.001) and the cumulative incidence of cardiac events.

Interpretation

Although the TIMI risk score is an effective risk stratification tool for patients in the emergency department with potential acute coronary syndromes, it should not be used as the sole means of determining patient disposition.Chest pain is a common presenting complaint in the emergency department that requires efficient risk stratification, timely initiation of treatment in high-risk patients and safe determination of patient disposition. Several studies have been published that stratify the risk of patients in the emergency department with chest pain.^1–5 However, only the Thrombolysis in Myocardial Infarction (TIMI) risk score, which was initially developed for use in patients with unstable angina or non–ST-segment elevation myocardial infarction or both,⁶ has been broadly validated in several independent emergency department populations with chest pain and thus constitutes the highest level of evidence available.The TIMI risk score assigns each of seven predictors a value of one point, allowing stratification of patients into one of eight prognostic categories (Box 1).⁶ The clinical end points are acute myocardial infarction, coronary revascularization and death from any cause.

Box 1.?Predictor variables included in the TIMI risk score^*

• Age of more than 65 years
• Three or more risk factors for atherosclerosis
• Known coronary artery disease
• Two or more episodes of anginal chest pain in the preceding 24 hours
• Acetylsalicylic acid use in the seven days before hospitalization
• ST-segment deviation of 0.05 mV or more
• Elevated cardiac markers

A robust estimate of the performance of the TIMI risk score obtained from a systematic review may prove useful to both clinicians and researchers. Clinicians would have a reliable quantitative estimate of a patient’s short-term risk of a cardiac event. This could be used as an adjunct to clinical acumen and as a tool to communicate risk to patients in a shared decision-making model of care.⁷ Researchers would also have an estimate of the prognostic accuracy of the TIMI risk score derived from different practice settings and patient populations that represent a wide variety of ethnic backgrounds. This estimate may serve as a useful baseline for comparison as emerging clinical prediction rules and imaging modalities continue to refine our approach to diagnosis and risk stratification in patients in the emergency department with potential acute coronary syndromes.We conducted a comprehensive systematic review and meta-analysis to assess the methodological quality and prognostic performance of studies that had prospectively validated the TIMI risk score in patients in the emergency department.     

Analysis and simulation of hollow-fiber bioreactor dynamics

A flexible and economical computer simulation model for the process analysis, parameter prediction, and optimal design of hollow-fiber biochemical reactors (HFBRs) has been developed. The validity and predictive capability of the HFBR simulator was successfully tested with two independent laboratory case studies. Of particular interest are the transient transport and bioconversion mechanisms including the effect of radial mass convection on the substrate uptake in the lumen. The portable computer code is an efficient tool to aid in theoretical and experimental investigations. The underlying principles used for the model development are applicable to a broad family of (membrane) bioreactors.     

Pathogenic effects of Salmonella enterica subspecies enterica serovar Typhimurium on sprouting and growth of maize

The study was undertaken to understand effects and survival of S. enterica subspecies enterica serovar Typhimurium (S. Typhimurium), a zoonotic serovar, on maize seed germination and plant growth. All the four strains of S. enterica subspecies enterica serovar Typhimurium significantly reduced germination of maize seeds in sprouting plates as well as in soil. About > or =2.7x10(3) Salmonella cfu ml(-1) of soaking water, while > or =2.7x10(7) Salmonella cfu g(-1) soil were required to significantly inhibit germination of maize. Similar inhibition of germination could be observed using > or = 16 mg of bacteria free Salmonella cell lysate (CL) protein per g of soil or > or =0.5 mg of CL protein per ml of soaking water in sprouting plates. At the constant dose of 3.6x10(7) to 3.8x10(7) Salmonella cfu or 5 mg cell lysate protein ml(-1) of soaking water, four strains of Salmonella significantly reduced germination, however difference between strains was insignificant. After germination too, maize growth was affected both by Salmonella organism and CL with little strain-to-strain variation. All Salmonella persisted in growing plants from 15 to 35 days of plant age and up to 190 days in soil. Maize plants once grown for a week in sterile soil were resistant to invasion of S. enterica subspecies enterica serovar Typhimurium in their leaves even in doses as high as 7.6x10(9) cfu g(-1) of soil. Salmonella persisted better and longer in plants grown from contaminated seed sown in loam soil, but rarely in plants grew in sandy soil. All maize plants had Salmonella in their stumps even after 35 days of sowing irrespective of kind of soil, primary source of infection (soil or seed) and type of S. enterica subspecies enterica serovar Typhimurium strain. The study revealed that Salmonella is not only zoonotic but a phytopathogen also.     

Cytotoxic factor-autoantibodies: possible role in the pathogenesis of dengue haemorrhagic fever

During dengue virus infection a unique cytokine, cytotoxic factor (hCF), is produced that is pathogenesis-related and plays a key role in the development of dengue haemorrhagic fever (DHF). However, what regulates the adverse effects of hCF is not known. We have previously shown that anti-hCF antibodies raised in mice, neutralise the pathogenic effects of hCF. In this study we have investigated the presence and levels of hCF-autoantibodies in sera of patients with various severity of dengue illness (n=136) and normal healthy controls (n=50). The highest levels of hCF-autoantibodies (mean+/-S.D.=36+/-20 U ml(-1)) were seen in patients with mild illness, the dengue fever (DF), and 48 out of 50 (96%) of the sera were positive. On the other hand the hCF-autoantibody levels declined sharply with the development of DHF and the levels were lowest in patients with DHF grade IV (mean+/-S.D.=5+/-2 U ml(-1); P=<0.001 as compared to DF). Only one of the 13 DHF grade IV patients had an antibody level above the 'cut-off' value (mean plus 3 S.D. of the control sera). The analysis of data with respect to different days of illness further showed that the highest levels of hCF-autoantibodies were present in DF patients at >9 days of illness. Moreover, the DF patients at all time points, i.e. 1-4, 5-8 and >9 days of illness had significantly higher levels of hCF-autoantibodies (P<0.001) than patients with DHF grade I, II, III and IV. In addition DHF grade I and grade II patients had significantly more positive specimens than DHF grade III and grade IV patients at all time points. These results suggest that elevated levels of hCF-autoantibodies protect the patients against the development of severe forms of DHF and, therefore, it may be useful as a prognostic indicator.