Biochemical characterization of Arabidopsis thaliana starch branching enzyme 2.2 reveals an enzymatic positive cooperativity.

Starch Branching Enzymes (SBE) catalyze the formation of α(1 â†’ 6) branching points on starch polymers: amylopectin and amylose. SBEs are classified in two groups named type 1 and 2. Both types are present in the entire plant kingdom except in some species such as Arabidopsis thaliana that expresses two type 2 SBEs: BE2.1 and BE2.2. The present work describes in vitro enzymatic characterization of the recombinant BE2.2. The function of recombinant BE2.2 was characterized in vitro using spectrophotometry assay, native PAGE and HPAEC-PAD analysis. Size Exclusion Chromatography separation and SAXS experiments were used to identify the oligomeric state and for structural analysis of this enzyme. Optimal pH and temperature for BE2.2 activity were determined to be pH 7 and 25 °C. A glucosyl donor of at least 12 residues is required for BE2.2 activity. The reaction results in the transfer in an α(1 â†’ 6) position of a glucan preferentially composed of 6 glucosyl units. In addition, BE2.2, which has been shown to be monomeric in absence of substrate, is able to adopt different active forms in presence of branched substrates, which affect the kinetic parameters. BE2.2 has substrate specificity similar to those of the other type-2 BEs. We propose that the different conformations of the enzyme displaying more or less affinity toward its substrates would explain the adjustment of the kinetic data to the Hill equation. This work describes the enzymatic parameters of Arabidopsis BE2.2. It reveals for the first time conformational changes for a branching enzyme, leading to a positive cooperative binding process of this enzyme.

Poor nutritional status is associated with other geriatric domain impairments and adverse postoperative outcomes in onco-geriatric surgical patients - A multicentre cohort study.

BACKGROUND: Nutritional status (NS), though frequently affected in onco-geriatric patients, is no standard part of a geriatric assessment. The aim of this study was to analyse the association between a preoperatively impaired NS and geriatric domain impairments and adverse postoperative outcomes in onco-geriatric surgical patients. METHODS: 309 patients ≥70 years undergoing surgery for solid tumours were prospectively recruited. Nine screening tools were preoperatively administered as part of a geriatric assessment. NS was based on BMI, weight loss and food intake. Odds ratio's (OR) and 95% confidence intervals (95% CI) were estimated using logistic regression analysis. The occurrence of 30-day adverse postoperative outcomes was recorded. RESULTS: At a median age of 76 years, 107 patients (34.6%) had an impaired NS. Decreased performance status and depression were associated with an impaired NS, when adjusted for tumour characteristics and comorbidities (ORPS>1 3.46; 95% CI 1.56-7.67. ORGDS>5 2.11; 95% CI 1.05-4.26). An impaired NS was an independent predictor for major complications (OR 3.3; 95% CI 1.6-6.8). Ten out of 11 patients who deceased had an impaired NS. CONCLUSION: An impaired NS is prevalent in onco-geriatric patients considered to be fit for surgery. It is associated with decreased performance status and depression. An impaired NS is a predictor for adverse postoperative outcomes. NS should be incorporated in a geriatric assessment.

Costs associated with emergency care and hospitalization for severe hypoglycemia.

BACKGROUND AND AIMS: We aimed to determine the direct economic cost of the management of severe hypoglycemia among people with diabetes in Italy. METHODS AND RESULTS: Data of cases with an acceptance diagnosis of hypoglycemia between January 2011 and June 2012 were collected in 46 Emergency Departments (EDs). Emergency care costs were computed by estimating the average cost per ambulance service, ED visit and short-term (<24 h) observation period. Hospitalization expenditure was estimated using the average cost reimbursed by the Italian healthcare system for hospital admission per patient with diabetes in a specific hospital ward. We retrieved 3516 hypoglycemic episodes occurring in subjects with diabetes. Half the cases (51.8%) required referral to EDs by means of the emergency ambulance services. A total of 1751 cases (49.8%) received an ED visit followed by discharge; 604 cases (17.2%) received a short-term observation period; 1161 (33.1%) were hospitalized. Unit costs for emergency care management were estimated at €205 for an ambulance call, €23 for an ED visit, and €220 for a short-term observation. The mean hospitalization cost was estimated at €5317; the average cost per each severe hypoglycemic event totaled €1911. From a base case assumption, the total direct cost of severe hypoglycemia in patients with diabetes in Italy was estimated to be approximately €23 million per year. CONCLUSION: Severe hypoglycemia in patients with diabetes constitutes a remarkable economic burden for national healthcare systems. Measures for preventing hypoglycemia are mandatory in diabetes management programs considering the impact on patients and on health spending.

The management of severe hypoglycemia by the emergency system: the HYPOTHESIS study.

BACKGROUND AND AIMS: Severe hypoglycemia is not rare in diabetes and markedly impacts on health resource use. We aimed to describe the characteristics of patients attending emergency departments (EDs) following a severe episode of hypoglycemia, the factors associated with the management of events and the final outcome. METHODS AND RESULTS: We carried out a retrospective analysis of cases attending 46 Italian EDs for hypoglycemia from January 2011 to June 2012. A total of 3753 records were retrieved from the databases of the participating centers, part of a network repeatedly involved in collaborative studies; 3516 episodes occurred in subjects with diabetes (median age, 76 years; range, 1-102). Comorbidities were recorded in 2320 (65.9%) diabetes cases; association with trauma or road accidents in 287 (8.2%) and 47 (1.3%), respectively. Patients were treated with insulin (49.8%), oral agents (31.4%), or combination treatment (15.1%). The event required assistance by the out-of-hospital Emergency services in 1821 cases (51.8%). Following the ED visit, admission to hospital departments was deemed necessary in 1161 cases (33.1%). Diabetes treatment (oral agents: OR, 1.63; 95% confidence interval (CI), 1.37-1.94), increasing age (OR, 1.39; 95% CI, 1.31-1.48) and the number of comorbidities (OR, 1.51; 95% CI, 1.38-1.66) were the main drivers of admission. The in-hospital death rate was 10%, associated with the number of comorbidities (OR, 1.28; 95%CI, 1.01-1.63). CONCLUSION: Severe hypoglycemia requiring referral to EDs is associated with a significant work-up of the Emergency services and a remarkable in-hospital death rate in frail individuals with diabetes.

Miniaturized fiber-optic ultrasound probes for endoscopic tissue analysis by micro-opto-mechanical technology.

A new Micro-Opto-Mechanical System (MOMS) technology for the fabrication of optoacoustic probes on optical fiber is presented. The technology is based on the thermoelastic emission of ultrasonic waves from patterned carbon films for generation and on extrinsic polymer Fabry-Perot acousto-optical transducers for detection, both fabricated on miniaturized single-crystal silicon frames used to mount the ultrasonic transducers on the tip of an optical fiber. Thanks to the fabrication process adopted, high miniaturization levels are reached in the MOMS devices, demonstrating fiber-optic emitters and detectors with minimum diameter around 350 and 250 µm respectively. A thorough functional testing of the ultrasound emitters mounted on 200 and 600 µm diameter optical fibers is presented, in which the fiber-optic emitter with a diameter of 200 µm shows generated acoustic pressures with peak-to-peak value up to 2.8 MPa with rather flat emission spectra extended beyond 150 MHz. The possibility to use the presented optoacoustic sources in conjunction with the fiber-optic acousto-optical detectors within a minimally invasive probe is also demonstrated by successfully measuring the ultrasonic echo reflected from a rigid surface immersed in water with various concentration of scatterers. The resulting spectra highlight the possibility to discriminate the effects due to frequency selective attenuation in a very wide range of frequencies within a biological medium using the presented fiber-optic probes.

SAXS conformational tracking of amylose synthesized by amylosucrases.

Amylose, a linear polymer of α(1,4)-linked glucosyl units and a major constituent of starch granules, can also be enzymatically synthesized in vitro from sucrose by bacterial amylosucrases. Depending on the initial sucrose concentration and the enzyme used, amylose oligomers (or polymers) are formed and self-associate during synthesis into various semicrystalline morphologies. This work describes for the first time a synchrotron SAXS study of the structure in solution of two amylosucrases, namely, NpAS and the thermostable DgAS, under conditions of polymer synthesis and, simultaneously, the amylose conformation. The structure in solution of both amylosucrases during the reaction was shown to be similar to the known crystallographic structures. The conformation of amylose produced at an early stage consists of a mixture of wormlike chains and double helical cylindrical structures. In the case of NpAS, in a second stage, individual double helices pack into clusters before crystallizing and precipitating. Amylose produced by DgAS never self-associates in such clusters due to the higher temperature used for amylose synthesis. All the dimensions determined for wormlike chains and cylindrical conformations at different times of NpAS synthesis are in very good agreement with structural features usually observed on gels of amylose extracted from starch. This provides new insights in understanding the mechanisms of amylose gelation.

Modification of anisotropic plasma diffusion via auxiliary electrons emitted by a carbon nanotubes-based electron gun in an electron cyclotron resonance ion source.

The diffusion mechanism in magnetized plasmas is a largely debated issue. A short circuit model was proposed by Simon, assuming fluxes of lost particles along the axial (electrons) and radial (ions) directions which can be compensated, to preserve the quasi-neutrality, by currents flowing throughout the conducting plasma chamber walls. We hereby propose a new method to modify Simon's currents via electrons injected by a carbon nanotubes-based electron gun. We found this improves the source performances, increasing the output current for several charge states. The method is especially sensitive to the pumping frequency. Output currents for given charge states, at different auxiliary electron currents, will be reported in the paper and the influence of the frequency tuning on the compensation mechanism will be discussed.

Rapid method for mean telomere length measurement directly from cell lysates.

Telomere length is involved in cell survival, tumorigenesis, and early aging. We present here an innovative method to determine the mean telomere length without any DNA purification. Our strategy is to measure both the DNA concentration and the number of telomeric units (TTAGGG) directly from cell lysate produced by the combined action of NaOH (pH>13) and sonication directly on cell pellet. Telomere units are quantified using an enzyme hybridization assay on 96-well microtiter plates grafted with a captor sequence. A biotin-coupled-tracer oligonucleotide hybridizes with telomere fragments and the enzymatic reaction is performed with a streptavidin-acetylcholinesterase conjugate, using the colorimetric method of Ellman. OD measure is directly proportional to the number of telomere units in cell lysate. This scalable technique allows the determination of mean telomere length simultaneously in many samples. This assay will be highly efficient to screen new drugs involved in chemotherapy targeting telomerase or directly telomeres.

Simple method for the quantitative analysis of tetracyclines by direct fluorimetry after thin-layer chromatography on cellulose plates.

A thin-layer chromatographic method is described in which tetracyclines are separated on a cellulose layer by development with aqueous solutions of certain salts (magnesium, calcium, barium and zinc chloride). The spots exhibit good fluorescence in UV light, allowing ready detection without the necessity for treatment with any reagents. This fluorescence can be used for the direct photometric determination of the tetracyclines with good accuracy.