Effect of Isovolemic,Isothermic Hemodialysis on Cerebral Perfusion and Vascular Stiffness Using Contrast Computed Tomography and Pulse Wave Velocity

Background

Patients undergoing hemodialysis treatment have a six-fold increased risk for stroke relative to the general population. However, the effect of hemodialysis on cerebral blood flow is poorly studied and confounding factors like blood pressure and ultrafiltration as well as temperature changes have rarely been accounted for. The aim of our study was to use state-of-the-art technology to evaluate the effect of a single dialysis session on cerebral perfusion as well as on vascular stiffness.

Methods

Chronic hemodialysis patients (7 male/3 female, mean age 58 years) were recruited. Cerebral blood flow and arterial pulse wave velocity were measured before and immediately after a hemodialysis session. To exclude effects of volume changes we kept ultrafiltration to a minimum, allowing no change in body weight. Isothermic conditions were maintained by using the GENIUS single-pass batch-dialysis system with a high-flux polysulfone dialyser. Cerebral blood flow was measured by contrast-enhanced computed tomography. Pulse wave velocity was measured using the SphygmoCor (AtCor Medical, USA) device by a single operator.

Results

This study shows for the first time that isovolemic, isothermic hemodialysis neither affected blood pressure or heart rate, nor total or regional cerebral perfusion. There was also no change in pulse wave velocity.

Conclusions

Mechanisms other than the dialysis procedure itself might be causative for the high incidence of ischemic strokes in this patient population. Moreover, the sole removal of uremic toxins does not lead to short-term effects on vascular stiffness, underlying the importance of volume control in this patient population.     

Development of a chemically defined medium for the production of enterolysin A from Enterococcus faecalis B9510

Aim

This study aimed to develop a simplified chemically defined medium that could sustain the growth and bacteriocin (enterolysin A) production by Enterococcus faecalis B9510.

Methods and Results

The nutritional requirements of E. faecalis B9510 in a chemically defined medium were determined by single omission experiments. It was observed that eight amino acids (arginine, glycine, histidine, isoleucine, leucine, methionine, tryptophan and valine), three B vitamins (nicotinic acid, Ca‐pantothenic acid and pyridoxal) and magnesium sulphate were essential for growth. Based on this information, a Simplified Defined Medium (SDM) was formed consisting of 26 components. Comparison of SDM with M‐17 showed that growth and bacteriocin production in SDM was similar to that in M‐17. The bacteriocin from SDM was then purified by ultrafiltration. The retentate of ultrafiltration step was analysed by SDS‐PAGE and the results showed a single active band in the gel, which was excised and analysed by mass spectrometry, which indicated that the active band was enterolysin A, a cell wall degrading bacteriocin.

Conclusions

A simplified defined medium can be formulated for the growth and bacteriocin production by Enterococcus faecalis, whose efficiency is comparable with that of a complex commercial medium.

Significance and Impact of the Study

The development of such a medium can be useful for bacteriocin production and subsequent purification in a simplified manner and, therefore, helpful in the identification of novel bacteriocins.     

Using a water-immiscible ionic liquid to improve asymmetric reduction of 4-(trimethylsilyl)-3-butyn-2-one catalyzed by immobilized Candida parapsilosis CCTCC M203011 cells

Background  

Whole cells are usually employed for biocatalytic reduction reactions to ensure efficient coenzyme regeneration and to avoid problems with enzyme purification and stability. The efficiency of whole cell-catalyzed bioreduction is frequently restricted by pronounced toxicity of substrate and/or product to the microbial cells and in many instances the use of two-phase reaction systems can solve such problems. Therefore, we developed new, biphasic reaction systems with biocompatible water-immiscible ionic liquids (ILs) as alternatives to conventional organic solvents, in order to improve the asymmetric reduction of 4-(trimethylsilyl)-3-butyn-2-one (TMSB) to (S)-4-(trimethylsilyl)-3-butyn-2-ol {(S)-TMSBOL}, a key intermediate for synthesis of 5-lipoxygenase inhibitors, using immobilized Candida parapsilosis CCTCC M203011 cells as the biocatalyst.     

Evaluation of transduction efficiency in macrophage colony-stimulating factor differentiated human macrophages using HIV-1 based lentiviral vectors

Background  

Monocyte-derived macrophages contribute to atherosclerotic plaque formation. Therefore, manipulating macrophage function could have significant therapeutic value. The objective of this study was to determine transduction efficiency of two HIV-based lentiviral vector configurations as delivery systems for the transduction of primary human blood monocyte-derived macrophages.     

Evaluation of the mass transfer rate using computer simulation in a three-dimensional interwoven hollow fiber-type bioartificial liver

Objectives

To determine the most efficient design of a hollow fiber-based bioreactor device for a bioartificial liver support system through comparative bioengineering evaluations.

Results

We compared two types of hollow fiber-based bioreactors, the interwoven-type bioreactor (IWBAL) and the dialyzer-type bioreactor (DBAL), by evaluating the overall mass transfer coefficient (K) and the convective coefficient (X). The creatinine and albumin mass transfer coefficients and convective coefficients were calculated using our mathematical model based on the homoporous theory and the modified Powell method. Additionally, using our model, we simulated the mass transport efficiency in clinical-scale BALs. The results of this experiment demonstrate that the mass transfer coefficients for creatinine and albumin increased proportionally with velocity with the IWBAL, and were consistently greater than that found with the DBAL. These differences were further enhanced in the simulation of the large-scale model.

Conclusions

Our findings indicate that the IWBAL with its unique 30° cross hollow fiber design can provide greater solute removal and more efficient metabolism when compared to the conventional DBAL design.

     

Effect of variable heat transfer coefficient on tissue temperature next to a large vessel during radiofrequency tumor ablation

Background  

One of the current shortcomings of radiofrequency (RF) tumor ablation is its limited performance in regions close to large blood vessels, resulting in high recurrence rates at these locations. Computer models have been used to determine tissue temperatures during tumor ablation procedures. To simulate large vessels, either constant wall temperature or constant convective heat transfer coefficient (h) have been assumed at the vessel surface to simulate convection. However, the actual distribution of the temperature on the vessel wall is non-uniform and time-varying, and this feature makes the convective coefficient variable.     

Active DNA demethylation in human postmitotic cells correlates with activating histone modifications,but not transcription levels

Background  

In mammals, the dynamics of DNA methylation, in particular the regulated, active removal of cytosine methylation, has remained a mystery, partly due to the lack of appropriate model systems to study DNA demethylation. Previous work has largely focused on proliferating cell types that are mitotically arrested using pharmacological inhibitors to distinguish between active and passive mechanisms of DNA demethylation.     

In vitro calibration of a system for measurement of in vivo convective heat transfer coefficient in animals

Background  

We need a sensor to measure the convective heat transfer coefficient during ablation of the heart or liver.     

Abrin Immunotoxin: Targeted Cytotoxicity and Intracellular Trafficking Pathway

Background

Immunotherapy is fast emerging as one of the leading modes of treatment of cancer, in combination with chemotherapy and radiation. Use of immunotoxins, proteins bearing a cell-surface receptor-specific antibody conjugated to a toxin, enhances the efficacy of cancer treatment. The toxin Abrin, isolated from the Abrus precatorius plant, is a type II ribosome inactivating protein, has a catalytic efficiency higher than any other toxin belonging to this class of proteins but has not been exploited much for use in targeted therapy.

Methods

Protein synthesis assay using ³[H] L-leucine incorporation; construction and purification of immunotoxin; study of cell death using flow cytometry; confocal scanning microscopy and sub-cellular fractionation with immunoblot analysis of localization of proteins.

Results

We used the recombinant A chain of abrin to conjugate to antibodies raised against the human gonadotropin releasing hormone receptor. The conjugate inhibited protein synthesis and also induced cell death specifically in cells expressing the receptor. The conjugate exhibited differences in the kinetics of inhibition of protein synthesis, in comparison to abrin, and this was attributed to differences in internalization and trafficking of the conjugate within the cells. Moreover, observations of sequestration of the A chain into the nucleus of cells treated with abrin but not in cells treated with the conjugate reveal a novel pathway for the movement of the conjugate in the cells.

Conclusions

This is one of the first reports on nuclear localization of abrin, a type II RIP. The immunotoxin mAb F1G4-rABRa-A, generated in our laboratory, inhibits protein synthesis specifically on cells expressing the gonadotropin releasing hormone receptor and the pathway of internalization of the protein is distinct from that seen for abrin.     

Sorting of cells of the same size, shape, and cell cycle stage for a single cell level assay without staining

Background  

Single-cell level studies are being used increasingly to measure cell properties not directly observable in a cell population. High-performance data acquisition systems for such studies have, by necessity, developed in synchrony. However, improvements in sample purification techniques are also required to reveal new phenomena. Here we assessed a cell sorter as a sample-pretreatment tool for a single-cell level assay. A cell sorter is routinely used for selecting one type of cells from a heterogeneous mixture of cells using specific fluorescence labels. In this case, we wanted to select cells of exactly the same size, shape, and cell-cycle stage from a population, without using a specific fluorescence label.     

Assessment of human adenovirus removal by qPCR in an advanced water reclamation plant in Georgia,USA

Aims

To assess human adenoviruses (HAdVs) removal in an advanced wastewater treatment facility and compare two parallel tertiary treatment methods for the removal of HAdVs.

Methods and Results

Tangential flow ultrafiltration was used to concentrate the water samples, and HAdVs were precipitated by polyethylene glycol. HAdVs were detected only by TaqMan real‐time PCR, and HAdV genotype was determined by DNA sequence. HAdVs were detected in 100% of primary clarification influent, secondary clarification effluent and granular media (GM) filtration effluent samples but only in 31·2% of membrane filtration (MF) effluent and 41·7% of final effluent (FE) samples, respectively. The average HAdVs loads were significantly reduced along the treatments but HAdVs were still present in FE. Comparison of two parallel treatments (GM vs MF) showed that MF was technically superior to GM for the removal of HAdVs.

Conclusions

These findings indicate that adenoviruses are not completely removed by treatment processes. MF is a better treatment for removal of adenoviruses than GM filtration. Because only qPCR was used, the results only indicate the removal of adenovirus DNA and not the infectivity of viruses.

Significance and Impact of the Study

Presence of HAdVs in FE by qPCR suggests a potential public health risk from exposure to the treated wastewater and using the FE for recreational or water reuse purposes should be cautious.     

Comprehensive comparative-genomic analysis of Type 2 toxin-antitoxin systems and related mobile stress response systems in prokaryotes

Background  

The prokaryotic toxin-antitoxin systems (TAS, also referred to as TA loci) are widespread, mobile two-gene modules that can be viewed as selfish genetic elements because they evolved mechanisms to become addictive for replicons and cells in which they reside, but also possess "normal" cellular functions in various forms of stress response and management of prokaryotic population. Several distinct TAS of type 1, where the toxin is a protein and the antitoxin is an antisense RNA, and numerous, unrelated TAS of type 2, in which both the toxin and the antitoxin are proteins, have been experimentally characterized, and it is suspected that many more remain to be identified.     

Column Chromatography and Cell Culture Assay of Pseudomonas aeruginosa Toxin Z Preparations

Toxic material produced by Pseudomonas aeruginosa in cell culture was concentrated and partially purified. This toxic material, designated toxin Z, was produced during the growth of strain PA Z or PA 103 in HEp-2 monolayer cultures using Eagle minimal essential medium with 10% serum. Toxin Z, concentrated fourfold by Lyphogel or ultrafiltration, was used to produce antiserum in rabbits and also was fractionated by column chromatography, Twentyfold purification of toxin Z was obtained on a Sephadex G-200 column. Toxic column fractions were confirmed to have toxin Z by neutralization with specific antiserum. During concentration, purification, and neutralization procedures, the toxin was assayed exclusively by the cytopathic effect it produced in cell culture.     

Schoolhouse wastewater purification in a LWA-filled hybrid constructed wetland in Estonia

This paper analyses the purification efficiency and mass removal of organic material, suspended solids, nitrogen and phosphorus in a hybrid constructed wetland (CW) system treating wastewater from a basic school in Paistu, Estonia. The CW consists of two subsurface flow filter beds using lightweight aggregates (LWA): a two-chamber vertical subsurface flow (VSSF) filter bed followed by a horizontal subsurface flow (HSSF) filter bed, with a total area of 432 m². This CW was constructed in summer 2002 by the Centre for Ecological Engineering in Tartu (CEET). Eighteen series of water samples (from 30.10.2003 to 15.10.2005) were undertaken. The analyses show the outstanding purification effect of the system: for BOD₇ the average purification efficiency is 91%; for total suspended solids (TSS)—78%, for total P—89%, for total N—63%, and for NH₄N—77%. The average outlet values for the above-listed parameters were 5.5, 7.0, 0.4, 19.2 and 9.1 mg L⁻¹, respectively. According to our results, the purification parameters meet the standards set by the Water Act of Estonia for wastewater treatment plants of 2000–9999 PE: 15, 25, and 1.5 mg L⁻¹ for BOD₇, TSS and total P, respectively. The results show that hybrid CW systems consisting of subsurface flow filter beds can work efficiently in conditions of changing hydraulic loading and relatively cold climate. We did not find significant differences between the removal efficiency, mass removal, and values of the first-order rate-constant k for most water quality indicators during the warm (May–October) and cold (November–April) periods. Locally produced LWA as a filter material in CWs has shown good hydraulic conductivity and phosphorus sorption capacity (k = 17.1 ± 12.4 m yr⁻¹). The Paistu CW, with its proper design and outstanding purification results, can be considered one of the best systems in Estonia.     

A Rapid and Economic In-House DNA Purification Method Using Glass Syringe Filters

Background

Purity, yield, speed and cost are important considerations in plasmid purification, but it is difficult to achieve all of these at the same time. Currently, there are many protocols and kits for DNA purification, however none maximize all four considerations.

Methodology/Principal Findings

We now describe a fast, efficient and economic in-house protocol for plasmid preparation using glass syringe filters. Plasmid yield and quality as determined by enzyme digestion and transfection efficiency were equivalent to the expensive commercial kits. Importantly, the time required for purification was much less than that required using a commercial kit.

Conclusions/Significance

This method provides DNA yield and quality similar to that obtained with commercial kits, but is more rapid and less costly.     

Simplified screening for the detection of soluble fusion constructs expressed in <Emphasis Type="Italic">E. coli</Emphasis> using a modular set of vectors

Background  

The solubility of recombinant proteins expressed in bacteria is often disappointingly low. Several strategies have been developed to improve the yield and one of the most common strategies is the fusion of the target protein with a suitable partner. Despite several reports on the successful use of each of these carriers to increase the solubility of some recombinant proteins, none of them was always successful and a combinatorial approach seems more efficient to identify the optimal combination for a specific protein. Therefore, the efficiency of an expression system critically depends on the speed in the identification of the optimal combination for the suitable fusion candidate in a screening process. This paper describes a set of expression vectors (pETM) designed for rapid subcloning, expression and subsequent purification using immobilized metal affinity chromatography (IMAC).     

Catechin-mediated restructuring of a bacterial toxin inhibits activity

Background

Catechins, polyphenols derived from tea leaves, have been shown to have antibacterial properties, through direct killing of bacteria as well as through inhibition of bacterial toxin activity. In particular, certain catechins have been shown to have bactericidal effects on the oral bacterium, Aggregatibacter actinomycetemcomitans, as well as the ability to inhibit a key virulence factor of this organism, leukotoxin (LtxA). The mechanism of catechin-mediated inhibition of LtxA has not been shown.

A new enzymatic route for production of long 5'-phosphorylated oligonucleotides using suicide cassettes and rolling circle DNA synthesis

Background  

The quality of chemically synthesized oligonucleotides falls with the length of the oligonucleotide, not least due to depurinations and premature termination during production. This limits the use of long oligonucleotides in assays where long high-quality oligonucleotides are needed (e.g. padlock probes). Another problem with chemically synthesized oligonucleotides is that secondary structures contained within an oligonucleotide reduce the efficiency of HPLC and/or PAGE purification. Additionally, ligation of chemically synthesized oligonucleotides is less efficient than the ligation of enzymatically produced DNA molecules.     

Uptake and biotransformation of pure commercial microcystin-LR versus microcystin-LR from a natural cyanobacterial bloom extract in the aquatic fungus <Emphasis Type="Italic">Mucor hiemalis</Emphasis>

Objectives

To evaluate the remediation efficiency of Mucor hiemalis by comparing media elimination, uptake, and biotransformation of microcystin-LR with exposure to pure toxin versus a crude bloom extract.

Results

With exposure to the extract, the elimination rate of microcystin-LR from the media, which was 0.28 ng MC-LR l^?1 h^?1, was significantly higher compared to that achieved with exposure to the pure toxin (0.16 ng MC-LR l^?1 h^?1) after 24 h. However, intracellular breakdown of microcystin-LR was significantly lower in the extract exposed pellets compared to the pure toxin treated fungal pellets over time. This coincided with reduced intracellular glutathione S-transferase activity with crude extract exposure which could be responsible for the detection of only the glutathione conjugate of microcystin-LR.

Conclusion

This paper signifies the importance of using laboratory exposure scenarios which resemble conditions in nature to fully understand and evaluate remediation efficiency. There is merit in using M. hiemalis for mycoremediation of cyanotoxins in surface waters.