Ammonium accumulation is associated with senescence of rice leaves

The relationship between ammonium accumulation and senescence of detached rice leaves was investigated. Ammonium accumulation in detached rice leaves coincided closely with dark-induced senescence. Exogenous NH4Cl and methionine sulfoximine, which caused an accumulation of ammonium in detached rice leaves, promoted senescence. Treatments such as light and benzyladenine, which retarded senescence, decreased ammonium level in detached rice leaves. Abscisic acid, which promoted senescence, increased ammonium level in detached rice leaves. The current results suggest that ammonium accumulation may be involved in regulating senescence. Evidence was presented to show that ammonium accumulated in detached rice leaves increases tissue sensitivity to ethylene. The accumulation of ammonium in detached rice leaves during dark-induced senescence is attributed to a decrease in glutamine synthetase activity and an increase in reduction of nitrate.     

Rapid purification of recombinant listeriolysin O (LLO) from Escherichia coli

Listeria monocytogenes is an emerging foodborne pathogen that is responsible for about 28% of the food-related deaths in the United States. It causes meningitis, septicaemia and in pregnant women, abortions and stillbirths. It secretes the toxin listeriolysin O (LLO) that allows the bacteria to enter the cytoplasm of host cells, where they can replicate and cause further infection. The rapid and sensitive detection of LLO in food samples is a key to monitoring and prevention of listeriosis. To facilitate the development of an assay for the specific detection of LLO, a source of LLO is essential. We outline a method of producing a large amount of functional LLO by expressing the hlyA gene (encoding LLO) in Escherichia coli and purifying the recombinant LLO using a one-step purification method. Purification of the protein takes only about 4 h. We compared three different expression constructs for the production of the toxin, which tends to interact strongly with a number of column surfaces. The first construct, using an intein fusion system, could not be purified from the column. The second LLO construct contained an N-terminus His tag; it gave a yield of 3.5–8 mg l⁻¹. The third contained a C-terminus His tag; it gave a yield of 2.5 mg l⁻¹ LLO. The purified LLO from the latter two constructs retained its activity at 4°C for over a year as determined by bovine red blood cell hemolysis assay. This paper provides a much-needed, high-yield, one-step purification method of recombinant LLO, and is the first to provide evidence of long-term stability of the toxin for further applications.     

Membrane lysis by the antibacterial peptides cecropins B1 and B3: A spin-label electron spin resonance study on phospholipid bilayers

Custom antibacterial peptides, cecropins B1 (CB1) and B3 (CB3), were synthesized. These peptides have particular sequence characteristics, with CB1 having two amphipathic alpha-helical segments and CB3 having two hydrophobic alpha-helical segments. These differences were exploited for a study of their efficacy in breaking up liposomes, which had different combinations of phosphatidic acid (PA) and phosphatidylcholine (PC), and a study of their lipid binding ability. Binding and nonbinding lysis actions of CB1 and CB3 on liposomes were examined further by electron spin resonance (ESR). The spin-labeled lipids 5'SL-PC, 7'SL-PC, 10'SL-PC, 12'SL-PC, and 16'SL-PC were used as probes. The ESR spectra revealed larger outer hyperfine splittings (2A(max)) for CB1 when the interactions of CB1 and CB3 with liposomes were compared. These observations indicate a larger restriction of the motion of the spin-labeled chains in the presence of CB1. Plots of the effective order parameter at the various probe positions (chain flexibility gradient) versus the peptide-lipid ratio further suggested that the lysis action of CB1 is related to its capacity to bind to the lipid bilayers. In contrast, there is no evidence of binding for CB3. To augment these findings, four spin-labeled peptides, C8SL-CB1, C32SL-CB1, C5SL-CB3, and C30SL-CB3, were also examined for their binding to and their state of aggregation within the lipid bilayers. Association isotherms of the peptides were measured for liposomes containing two molar fractions of PA (0.25 and 0.75). The membrane binding of the CB1 peptides exhibited a cooperative behavior, whereas the association isotherm of CB3 revealed binding to the lipid only for beta = 0.75 liposomes. To further identify the location of CB1 in the lipid bilayers, measurements of the collision rate with chromium oxalate in solution were conducted. Results from ESR power saturation measurements suggested that the NH(2)-terminal alpha-helix of CB1 is located on the surface of the lipid bilayers, whereas the COOH-terminal alpha-helix of CB1 is embedded below the surface of the lipid bilayers. These conclusions were further supported by the observed relationship between the partition distribution of peptides bound to liposomes at different PA/PC ratios and the amounts of free peptides. Based on the above observations, possible mechanisms of the bilayer lysis induced by CB1 and CB3 on liposomes of different composition are discussed.     

Use of the SAMe-TT2R2 Score to Predict Good Anticoagulation Control with Warfarin in Chinese Patients with Atrial Fibrillation: Relationship to Ischemic Stroke Incidence

Background

The efficacy and safety of warfarin therapy for stroke prevention in atrial fibrillation (AF) depends on the time in therapeutic range (TTR). We aimed to assess the predictive ability of SAMe-TT₂R₂ score in Chinese AF patients on warfarin, whose TTR is notoriously poor.

Methods and Results

This is a single-centre retrospective study. Patients with non-valvular AF on warfarin diagnosed between 1997 and 2011 were stratified according to SAMe-TT₂R₂ score, and TTR was calculated using Rosendaal method. The predictive power of SAMe-TT₂R₂ scores for good TTR i.e. >70% was assessed. We included 1,428 Chinese patients (mean age 76.2±8.7 years, 47.5% male) with non-valvular AF on warfarin. The mean and median TTR were 38.2±24.4% and 38.8% (interquartile range: 17.9% and 56.2%) respectively. TTR decreased progressively with increasing SAMe-TT₂R₂ score (p = 0.016). When the cut-off value of SAMe-TT₂R₂ score was set to 2, the sensitivity and specificity to predict TTR<70% were 85.7% and 17.8%, respectively. The corresponding positive and negative predictive values were 10.1% and 92.0%. After a mean follow-up of 4.7±3.6 years, 338 patients developed an ischemic stroke (4.96%/year). Patients with TTR≥70% had a lower annual risk of ischemic stroke of 3.67%/year compared with than those with TTR<70% (5.13%/year)(p = 0.08). Patients with SAMe-TT₂R₂ score ≤2 had the lowest risk of annual risk of ischemic stroke (3.49%/year) compared with those with SAMe-TT₂R₂ score = 3 (4.56%/year), and those with SAMe-TT₂R₂ score ≥4 (6.41%/year)(p<0.001). There was also a non-significant trend towards more intracranial hemorrhage with increasing SAMe-TT₂R₂ score.

Conclusions

The SAMe-TT₂R₂ score correlates well with TTR in Chinese AF patients, with a score >2 having high sensitivity and negative predictive values for poor TTR. Ischemic stroke risk increased progressively with increasing SAMe-TT₂R₂ score, consistent with poorer TTRs at high SAMe-TT₂R₂ scores.     

Structure-function analysis with tissue-type plasminogen activator. Effect of deletion of NH2-terminal domains on its biochemical and biological properties

Tissue-type plasminogen activator (t-PA) is a mosaic protein containing several distinct structural domains attached to the serine protease catalytic unit present at its COOH terminus. To investigate structure-function relationships in t-PA, we deleted the NH2-terminal domains, finger and epidermal growth factor, by genetic engineering. The genes for the parent and mutant t-PA were expressed in a bovine papilloma virus-dependent mammalian cell system. The secreted proteins were purified to homogeneity. The mutant protein was processed to the expected size of about 60 kDa compared to approximately 68 kDa for the parent t-PA, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fibrin autography. While the mutant t-PA had amidolytic activity comparable to native t-PA, it did not bind appreciably to fibrin. Consequently, fibrin-dependent enzymic activity, i.e. plasminogen activation in the presence of soluble fibrin and fibrinolysis were lower than with native recombinant t-PA. The effect of deletion of NH2-terminal domains on the plasma half-life (t1/2) was investigated by injecting native and mutant t-PA into mice. While the majority of the t-PA disappeared initially with a t1/2 of about 2 min, mutant t-PA cleared at a much slower rate with t1/2 of about 50 min. These findings suggest that the NH2-terminal domains of t-PA not only determine its specificity for binding to fibrin but also mediate its clearance from plasma in vivo. Furthermore, the catalytic unit in t-PA seems to function autonomously.     

Selective ectodomain phosphorylation and regulated cleavage of beta-amyloid precursor protein.

The beta-amyloid precursor protein (beta APP) is a highly conserved integral membrane protein expressed in most mammalian tissues and found at highest levels in the nervous system. Cerebral deposition of the amyloid beta-peptide (A beta), derived by proteolysis of beta APP, is an early and invariant feature of Alzheimer's disease. Protein phosphorylation by protein kinase C (PKC) has been found to regulate the metabolism of beta APP into nonamyloidogenic and amyloidogenic derivatives, but both the mechanism of these effects and the nature of beta APP phosphorylation are unknown. When labeled in vivo with [32P]orthophosphate, beta APP was phosphorylated only on serine residues in the N-terminal half of the extracellular domain, resulting in the secretion of phosphorylated soluble beta APP. PKC-mediated stimulation of beta APP secretion and concurrent inhibition of A beta release did not involve enhanced phosphorylation of beta APP and proceeded in the absence of cytoplasmic or extracellular phosphorylation of the precursor. The region of beta APP required for this indirect regulation by PKC was largely restricted to a 64 amino acid stretch around the secretory cleavage site. Moreover, in a truncated molecule designed to release soluble beta APP without the need for proteolytic cleavage, secretion was no longer regulated by PKC. Our data indicate that PKC-mediated pathways play a pivotal role in the control of beta APP metabolism and amyloid formation. However, in contrast to current postulates, this regulation is independent of beta APP phosphorylation and instead involves phosphorylation of other substrates that alter beta APP processing, such as beta APP-cleaving proteases.     

Elucidating the Role of a Tetrafluoroborate‐Based Ionic Liquid at the n‐Type Oxide/Perovskite Interface

Halide perovskites are currently one of the most heavily researched emerging photovoltaic materials. Despite achieving remarkable power conversion efficiencies, perovskite solar cells have not yet achieved their full potential, with the interfaces between the perovskite and the charge‐selective layers being where most recombination losses occur. In this study, a fluorinated ionic liquid (IL) is employed to modify the perovskite/SnO₂ interface. Using Kelvin probe and photoelectron spectroscopy measurements, it is shown that depositing the perovskite onto an IL‐treated substrate results in the crystallization of a perovskite film which has a more n‐type character, evidenced by a decrease of the work function and a shift of the Fermi level toward the conduction band. Photoluminescence spectroscopy and time‐resolved microwave conductivity are used to investigate the optoelectronic properties of the perovskite grown on neat and IL‐modified surfaces and it is found that the modified substrate yields a perovskite film which exhibits an order of magnitude lower trap density than the control. When incorporated into solar cells, this interface modification results in a reduction in the current–voltage hysteresis and an improvement in device performance, with the best performing devices achieving steady‐state PCEs exceeding 20%.     

Laboratory Test Surveillance following Acute Kidney Injury

Background

Patients with hospitalized acute kidney injury (AKI) are at increased risk for accelerated loss of kidney function, morbidity, and mortality. We sought to inform efforts at improving post-AKI outcomes by describing the receipt of renal-specific laboratory test surveillance among a large high-risk cohort.

Methods

We acquired clinical data from the Electronic health record (EHR) of 5 Veterans Affairs (VA) hospitals to identify patients hospitalized with AKI from January 1^st, 2002 to December 31^st, 2009, and followed these patients for 1 year or until death, enrollment in palliative care, or improvement in renal function to estimated GFR (eGFR) ≥60 L/min/1.73 m². Using demographic data, administrative codes, and laboratory test data, we evaluated the receipt and timing of outpatient testing for serum concentrations of creatinine and any as well as quantitative proteinuria recommended for CKD risk stratification. Additionally, we reported the rate of phosphorus and parathyroid hormone (PTH) monitoring recommended for chronic kidney disease (CKD) patients.

Results

A total of 10,955 patients admitted with AKI were discharged with an eGFR<60 mL/min/1.73 m². During outpatient follow-up at 90 and 365 days, respectively, creatinine was measured on 69% and 85% of patients, quantitative proteinuria was measured on 6% and 12% of patients, PTH or phosphorus was measured on 10% and 15% of patients.

Conclusions

Measurement of creatinine was common among all patients following AKI. However, patients with AKI were infrequently monitored with assessments of quantitative proteinuria or mineral metabolism disorder, even for patients with baseline kidney disease.     

Scoring Systems for Predicting Mortality after Liver Transplantation

Background

Liver transplantation can prolong survival in patients with end-stage liver disease. We have proposed that the Sequential Organ Failure Assessment (SOFA) score calculated on post-transplant day 7 has a great discriminative power for predicting 1-year mortality after liver transplantation. The Chronic Liver Failure - Sequential Organ Failure Assessment (CLIF-SOFA) score, a modified SOFA score, is a newly developed scoring system exclusively for patients with end-stage liver disease. This study was designed to compare the CLIF-SOFA score with other main scoring systems in outcome prediction for liver transplant patients.

Methods

We retrospectively reviewed medical records of 323 patients who had received liver transplants in a tertiary care university hospital from October 2002 to December 2010. Demographic parameters and clinical characteristic variables were recorded on the first day of admission before transplantation and on post-transplantation days 1, 3, 7, and 14.

Results

The overall 1-year survival rate was 78.3% (253/323). Liver diseases were mostly attributed to hepatitis B virus infection (34%). The CLIF-SOFA score had better discriminatory power than the Child-Pugh points, Model for End-Stage Liver Disease (MELD) score, RIFLE (risk of renal dysfunction, injury to the kidney, failure of the kidney, loss of kidney function, and end-stage kidney disease) criteria, and SOFA score. The AUROC curves were highest for CLIF-SOFA score on post-liver transplant day 7 for predicting 1-year mortality. The cumulative survival rates differed significantly for patients with a CLIF-SOFA score ≤8 and those with a CLIF-SOFA score >8 on post-liver transplant day 7.

Conclusion

The CLIF-SOFA score can increase the prediction accuracy of prognosis after transplantation. Moreover, the CLIF-SOFA score on post-transplantation day 7 had the best discriminative power for predicting 1-year mortality after liver transplantation.