Selective Permeation and Organic Extraction of Recombinant Green Fluorescent Protein (gfpuv) from Escherichia coli

Background Transformed cells of Escherichia coli DH5-α with pGFPuv, induced by IPTG (isopropyl-β-d-thiogalactopyranoside), express the green fluorescent protein (gfpuv) during growth phases. E. coli subjected to the combination of selective permeation by freezing/thawing/sonication cycles followed by the three-phase partitioning extraction (TPP) method were compared to the direct application of TPP to the same culture of E. coli on releasing gfpuv from the over-expressing cells. Material and Methods Cultures (37°C/100 rpm/ 24 h; μ = 0.99 h-1 - 1.10 h-1) of transformed (pGFP) Escherichia coli DH5-α, expressing the green fluorescent protein (gfpuv, absorbance at 394 nm and emission at 509 nm) were sonicated in successive intervals of sonication (25 vibrations/pulse) to determine the maximum amount of gfpuv released from the cells. For selective permeation, the transformed previously frozen (-75°C) cells were subjected to three freeze/thaw (-20°C/ 0.83°C/min) cycles interlaid by sonication (3 pulses/ 6 seconds/ 25 vibrations). The intracellular permeate with gfpuv in extraction buffer (TE) solution (25 mM Tris-HCl, pH 8.0, 1 mM β-mercaptoethanol β-ME, 0.1 mM PMSF) was subjected to the three-phase partitioning (TPP) method with t-butanol and 1.6 M ammonium sulfate. Sonication efficiency was verified on the application to the cells previously treated by the TPP method. The intra-cell releases were mixed and eluted through methyl HIC column with a buffer solution (10 mM Tris-HCl, 10 mM EDTA, pH 8.0). Results The sonication maximum released amount obtained from the cells was 327.67 μg gfpuv/mL (20.73 μg gfpuv/mg total proteins – BSA), after 9 min of treatment. Through the selective permeation by three repeated freezing/thawing/sonication cycles applied to the cells, a close content of 241.19 μg gfpuv/mL (29.74 μg gfpuv/mg BSA) was obtained. The specific mass range of gfpuv released from the same cultures, by the three-phase partitioning (TPP) method, in relation to total proteins, was higher, between 107.28 μg/mg and 135.10 μg/mg. Conclusions The selective permeation of gfpuv by freezing/thawing/sonication followed by TPP separation method was equivalent to the amount of gfpuv extracted from the cells directly by TPP; although selective permeation extracts showed better elution through the HIC column.

Effect of the root canal final rinse protocols on the debris and smear layer removal and on the push-out strength of an epoxy-based sealer

The aim of the present study was to evaluate the efficacy of QMiX, SmearClear, and 17% EDTA for the debris and smear layer removal from the root canal and its effects on the push-out bond strength of an epoxy-based sealer by scanning electron microscopy (SEM). Forty extracted human canines (n = 10) were assigned to the following final rinse protocols: G1-distilled water (control), G2–17% EDTA, G3-SmearClear, and G4-QMiX. The specimens were submitted to a SEM analysis to evaluate the presence of debris and smear layer, respectively, in the apical or cervical segments. In sequence, forty extracted human maxillary canines with the root canals instrumented were divided into four groups (n = 10) similar to the SEM analysis study. After the filling with AH Plus, the roots were transversally sectioned to obtain dentinal slices. The specimens were submitted to a push-out bond strength test using an electromechanical testing machine. The statistical analysis for the SEM and push-out bond strength studies were performed using the Kruskal–Wallis and Dunn tests (α = 5%). There was no difference among the G2, G3, and G4 efficacy in removing the debris and smear layer (P > 0.05). The efficacy of these groups was superior to the control group. The push-out bond strength values of G2, G3, and G4 were superior to the control group. The ability to remove the debris and smear layer by SmearClear and QMiX was as effective as the 17% EDTA. The final rinse with these solutions promoted similar push-out bond strength values.

Effect of passive ultrassonic instrumentation as a final irrigation protocol on debris and smear layer removal

This study sought to evaluate the efficacy of passive ultrasonic irrigation (PUI) on removing the smear layer and debris from root dentin using scanning electron microscopy (SEM). Twenty-five bovine incisors were manually prepared and divided into three groups according to the final irrigation protocol: EDTA, final irrigation with 12 mL of 17% EDTA for 3 minutes followed by 5 mL of 2.5% NaOCl; EDTA=PUI, final flush with 4 mL of 17% EDTA and PUI for 30 seconds. These procedures were repeated three times to standardize the volume of the irrigant. Control group, after preparation, the specimens were irrigated only with 17 mL of 2.5% NaOCl. The roots were fractured and analyzed using SEM. The intragroup analysis revealed that the EDTA=PUI protocol removed a higher amount of debris at the cervical third (P 5 0.03). The intergroup analysis revealed that EDTA=PUI presented the lowest amount of debris at the cervical third (P 5 0.007). Smear layer scores were higher in the control group compared with the EDTA and EDTA=PUI groups, but only at the cervical third (P 50.02). None of the final irrigant protocols completely removed the smear layer and debris. EDTA=PUI only improved the removal of debris at the cervical third.

Does long-term creatine supplementation impair kidney function in resistance-trained individuals consuming a high-protein diet?

Abstract Background The aim of this study was to determine the effects of creatine supplementation on kidney function in resistance-trained individuals ingesting a high-protein diet. Methods A randomized, double-blind, placebo-controlled trial was performed. The participants were randomly allocated to receive either creatine (20 g/d for 5 d followed by 5 g/d throughout the trial) or placebo for 12 weeks. All of the participants were engaged in resistance training and consumed a high-protein diet (i.e., ≥ 1.2 g/Kg/d). Subjects were assessed at baseline (Pre) and after 12 weeks (Post). Glomerular filtration rate was measured by 51Cr-EDTA clearance. Additionally, blood samples and a 24-h urine collection were obtained for other kidney function assessments. Results No significant differences were observed for 51Cr-EDTA clearance throughout the trial (Creatine: Pre 101.42 ± 13.11, Post 108.78 ± 14.41 mL/min/1.73m2; Placebo: Pre 103.29 ± 17.64, Post 106.68 ± 16.05 mL/min/1.73m2; group x time interaction: F = 0.21, p = 0.64). Creatinine clearance, serum and urinary urea, electrolytes, proteinuria, and albuminuria remained virtually unchanged. Conclusions A 12-week creatine supplementation protocol did not affect kidney function in resistance-trained healthy individuals consuming a high-protein diet; thus reinforcing the safety of this dietary supplement. Trial registration ClinicalTrials.gov NCT01817673

Chitosan: effect of a new chelating agent on the microhardness of root dentin

The effect of solutions of 0.2% chitosan, 15% EDTA and 10% citric acid on the microhardness of root dentin was evaluated comparatively in this study. Thirteen sound human maxillary central incisors were selected and decoronated at the cementoenamel junction. Ten roots were set into rapid polymerization acrylic resin and the root/resin block was fitted to the cutting machine to obtain slices from the cervical third. The first slice was discarded and the second slice was divided into four quadrants. Each quadrant was used to construct a sample, so that 4 specimens were obtained from each root slice, being one for each chelating solution to be tested: 15% EDTA, 10% citric acid, 0.2% chitosan and distilled water (control). The specimens were exposed to 50 μL of the solution for 5 min, and then washed in distilled water. A microhardness tester (Knoop hardness) with a 10 g load was used for 15 s. Data were analyzed statistically by one-way ANOVA and Tukey-Kramer test (α=0.05). The other 3 roots had the canals instrumented and irrigated at the end of the biomechanical preparation with the test solutions, and then examined by scanning electron microscopy (SEM) for qualitative analysis. All solutions reduced the microhardness of root dentin in a way that was statistically similar to each other (p>0.05) but significantly different from the control (p>0.05). The SEM micrographs showed that the three solutions removed smear layer from the middle third of the root canal. In conclusion, 0.2% chitosan, 15% EDTA and 10% citric acid showed similar effects in reducing dentin microhardness.

A preliminary study of the antibacterial potential of cetylpyridinium chloride in root canals infected by E. faecalis

The aim of this preliminary study was to verify the antibacterial potential of cetylpyridinium chloride (CPC) in root canals infected by Enterococcus faecalis. Forty human maxillary anterior teeth were prepared and inoculated with E. faecalis for 60 days. The teeth were randomly assigned to the following groups: 1: Root canal preparation (RCP) + 0.1% CPC with positive-pressure irrigation (PPI, Conventional, NaviTip®); 2: RCP + 0.2% CPC PPI; 3: RCP + 2.5% NaOCl PPI; 4: RCP + 2.5% NaOCl with negative-pressure irrigation system (NPI, EndoVac®); 5: Positive control; and 6: Negative control. Four teeth of each experimental group were evaluated by culture and 4 by scanning electron microscopy (SEM). In all teeth, the root canals were dried and filled with 17% EDTA (pH 7.2) for 3 min for smear layer removal. Samples from the infected root canals were collected and immersed in 7 mL of Letheen Broth (LB), followed by incubation at 37°C for 48 h. Bacterial growth was analyzed by turbidity of culture medium and then observed with a UV spectrophotometer. The irrigating solutions were further evaluated for antimicrobial effect by an agar diffusion test.The statistical data were treated by means, standard deviation, Kruskal-Wallis test and analysis of variance. Significance level was set at 5%. The results showed the presence of E. faecalis after root canal sanitization. The number of bacteria decreased after the use of CPC. In the agar diffusion test, CPC induced large microbial inhibition zones, similar to 2% chlorhexidine and large than 2.5% NaOCl. In conclusion, cetylpyridinium chloride showed antibacterial potential in endodontic infection with E. faecalis.

Dynamics of follicular growth and progesterone concentrations in cyclic and anestrous suckling Nelore cows (Bos indicus) treated with progesterone, equine chorionic gonadotropin, or temporary calf removal

The objective of this study was to investigate the effects of eCG and temporary calf removal (TCR) associated with progesterone (P4) treatment on the dynamics of follicular growth, CL size, and P4 concentrations in cyclic (n ¼ 36) and anestrous (n ¼ 30) Nelore cows. Cyclic (C) and anestrous (A) cows were divided into three groups. The control group received 2 mg of estradiol benzoate via intramuscular (IM) injection and an intravaginal device containing 1.9 g of P4 on Day 0. On Day 8, the device was removed, and the animals received 12.5 mg of dinoprost tromethamine IM. After 24 hours, the animals received 1 mg of estradiol benzoate IM. In the eCG group, cows received the same treatment described for the control group but also received 400 UI of eCG at the time of device removal. In the TCR group, calves were separated from the cows for 56 hours after device removal. Ultrasound exams were performed every 24 hours after device removal until the time of ovulation and 12 days after ovulation to measure the size of the CL. On the same day as the CL measurement, blood was collected to determine the plasma P4 level. Statistical analyses were performed with a significance level of P ≤ 0.05. In cyclic cows, the presence of the CL at the beginning of protocol resulted in a smaller follicle diameter at the time of device removal (7.4 ± 0.3 mm in cows with CL vs. 8.9 ± 0.4 mm in cows without CL; P ¼ 0.03). All cows ovulated within 72 hours after device removal. Anestrous cows treated with eCG or TCR showed follicle diameter at fixed-timed artificial insemination (A-eCG 10.2 ± 0.3 and A-TCR 10.3 ± 0.5 mm) and follicular growth rate (A-eCG 1.5 ± 0.2 and A-TCR 1.3 ± 0.1 mm/day) similar to cyclic cows (C-eCG 11.0 ± 0.6 and C-TCR 12.0 ± 0.5 mm) and (C-eCG 1.4 ± 0.2 and C-TCR 1.6 ± 0.2 mm/day, respectively; P ≤ 0.05). Despite the similarities in CL size, the average P4 concentration was higher in the A-TCR (9.6 ± 1.4 ng/mL) than in the A-control (4.0 ± 1.0 ng/mL) and C-TCR (4.4 ± 1.0 ng/mL) groups (P < 0.05). From these results, we conclude that eCG treatment and TCR improved the fertility of anestrous cows by providing follicular growth rates and size of dominant follicles similar to cyclic cows. Additionally, TCR increases the plasma concentrations of P4 in anestrous cows

Entwicklung neuer Biomarker zur Detektion von Umweltbelastung

Es wurde bis heute meist mit relativ aufwendigen Methoden nach dem Vorkommen von Strangbrüchen in der DNA gesucht. Die hier verwendete Fast-Micromethod steht jetzt neben mehreren bereits etablierten Methoden zur qualitativen und quantitativen Erfassung von DNA-Strangbrüchen zur Verfügung. Die Methode wurde zur schnellen und empfindlichen Messung von DNA-Schäden und deren Reparatur unter Verwendung von PicoGreen, einem spezifisch an DNA-Einzelstränge bindenden Fluoreszenzfarbstoff, durchgeführt. Die Methode wurde in der vorliegenden Arbeit für verschiedene Zellarten wie HeLa-Zellen und Schwamm-Zellen mit Schwermetallionen verwendet. Sie wurde ebenfalls bei verschiedenen Strangbruch-induzierenden Stoffen wie NQO und physikalischen Faktoren wie UV-Bestrahlung und Gammastrahlung erfolgreich eingesetzt. Die Strangbrüche wurden bei HeLa-Zellen mit 10 µM NQO nach 90 Minuten Inkubation und ebenso bei Gammastrahlung mit 16 Gy gemessen. An Schwammzellen wurden Effekte von Schwermetallionen und UV, auch einschließlich nachgeschalteter Reparaturaktivität, gemessen. Dabei war die Reparatur jedoch nicht ganz vollständig. Durch die Fast-Micromethod wurde DNA-Schädigung durch UV-Bestrahlung und Gentoxizität von Schwermetall-Ionen an HeLa-Zellen nachgewiesen. Die Schwammzellen wurden mit verschiedenen Schwermetall-Ionen in verschiedenen Konzentrationen getestet und mit UV bei verschiedenen Dosen bestrahlt. Teilweise wurde nach dem Einfluss die DNA-Reparatur erfasst. Schwammzellen wurden in den drei häufig verwendeten Medien CMFSW, CMFSW+EDTA und Seewasser aufgenommen. Im Seewasser waren die Zellen nach 60 Minuten Inkubationszeit aggregiert (Primmorphe). Im CMFSW+EDTA Medium lagen dagegen nur Einzellen vor, da die Zellen im EDTA-Medium dissoziiert wurden und auch nicht mehr proliferierten.

Characterization of major zinc containing myonecrotic and procoagulant metalloprotease 'malabarin' from non lethal trimeresurus malabaricus snake venom with thrombin like activity: its neutralization by chelating agents

A major myonecrotic zinc containing metalloprotease 'malabarin' with thrombin like activity was purified by the combination of gel permeation and anion exchange chromatography from T. malabaricus snake venom. MALDI-TOF analysis of malabarin indicated a molecular mass of 45.76 kDa and its N-terminal sequence was found to be Ile-Ile-Leu- Pro(Leu)-Ile-Gly-Val-Ile-Leu(Glu)-Thr-Thr. Atomic absorption spectral analysis of malabarin raveled the association of zinc metal ion. Malabarin is not lethal when injected i.p. or i.m. but causes extensive hemorrhage and degradation of muscle tissue within 24 hours. Sections of muscle tissue under light microscope revealed hemorrhage and congestion of blood vessel during initial stage followed by extensive muscle fiber necrosis with elevated levels of serum creatine kinase and lactate dehydrogenase activity. Malabarin also exhibited strong procoagulant action and its procoagulant action is due to thrombin like activity; it hydrolyzes fibrinogen to form fibrin clot. The enzyme preferentially hydrolyzes A? followed by B subunits of fibrinogen from the N-terminal region and the released products were identified as fibrinopeptide A and fibrinopeptide B by MALDI. The myonecrotic, fibrinogenolytic and subsequent procoagulant activities of malabarin was neutralized by specific metalloprotease inhibitors such as EDTA, EGTA and 1, 10-phenanthroline but not by PMSF a specific serine protease inhibitor. Since there is no antivenom available to neutralize local toxicity caused by T. malabaricus snakebite, EDTA chelation therapy may have more clinical relevance over conventional treatment.

Detection of ethyl glucuronide in blood spotted on different surfaces

This study aims to show that sensitive detection of ethyl glucuronide in dried blood spotted onto various surfaces after a period of 24h is feasible. At present, there is insufficient information how tightly ethyl glucuronide (EtG) binds to various materials and how easily it can be eluted. 4ml aliquots of blood samples obtained from seven volunteers after consumption of alcoholic beverages were applied to six different surfaces. After drying and a 24h-storage at 20±2°C the samples were re-dissolved in water, and EtG was subsequently analyzed by a LC-MS Paul-type ion trap. A comparison was made between dried and corresponding fluid samples. EtG was detectable in all subjects' samples following consumption of alcohol. EtG was also detectable after a storage time of four weeks at 4°C in whole blood that had been preserved with EDTA. EtG was detectable in all samples dried on different surfaces and its concentration remained relatively constant irrespective of the particular condition of the material. Detection of EtG in blood spots from the scene may indicate recent alcohol consumption in cases where collection of blood remained undone or could not be performed.

Demographics of blood pressure and hypertension in children on renal replacement therapy in Europe

Hypertension is a well-known complication in children on renal replacement therapy and an important risk factor for cardiovascular disease in later life. In order to define the prevalence of and risk factors for hypertension among children, we enrolled 3337 pediatric patients from 15 countries in the ESPN/ERA-EDTA Registry of whom 464 were on hemodialysis, 851 on peritoneal dialysis, and 2023 had received a renal allograft. Hypertension was defined as either systolic or diastolic blood pressures in the 95th percentile or greater for age, height, and gender or use of antihypertensive medication. Analyses were adjusted for age, gender, duration, and modality of renal replacement therapy. In 10 countries in which information on the use of antihypertensive medication was available, hypertension was present in over two-thirds of hemodialysis, peritoneal dialysis, or transplant patients. Blood pressure values above the 95th percentile were significantly more prevalent in very young patients (under 3 years) compared to 13- to 17-year olds (odds ratio 2.47), during the first year compared to over 5 years of renal replacement therapy (odds ratio 1.80), and in patients on hemodialysis compared to transplant recipients or those on peritoneal dialysis (odds ratios of 2.48 and 1.59, respectively). Over time, mean blood pressures decreased in both hemodialysis and transplant patients, but not in peritoneal dialysis patients. Hence, our findings highlight the extent of the problem of hypertension in children with end-stage renal disease in Europe.

Enamel matrix protein adsorption to root surfaces in the presence or absence of human blood

Background: The clinical use of an enamel matrix derivative (EMD) has been shown to promote formation of new cementum, periodontal ligament (PDL), and bone and to significantly enhance the clinical outcomes after regenerative periodontal surgery. It is currently unknown to what extent the bleeding during periodontal surgery may compete with EMD adsorption to root surfaces. The aim of this study is to evaluate the effect of blood interactions on EMD adsorption to root surfaces mimicking various clinical settings and to test their ability to influence human PDL cell attachment and proliferation. Methods: Teeth extracted for orthodontic reasons were subjected to ex vivo scaling and root planing and treated with 24% EDTA, EMD, and/or human blood in six clinically related settings to determine the ability of EMD to adsorb to root surfaces. Surfaces were analyzed for protein adsorption via scanning electron microscopy and immunohistochemical staining with an anti-EMD antibody. Primary human PDL cells were seeded on root surfaces and quantified for cell attachment and cell proliferation. Results: Plasma proteins from blood samples altered the ability of EMD to adsorb to root surfaces on human teeth. Samples coated with EMD lacking blood demonstrated a consistent even layer of EMD adsorption to the root surface. In vitro experiments with PDL cells demonstrated improved cell attachment and proliferation in all samples coated with EMD (irrespective of EDTA) when compared to samples containing human blood. Conclusion: Based on these findings, it is advised to minimize blood interactions during periodontal surgeries to allow better adsorption of EMD to root surfaces.

Wound healing following regenerative procedures in furcation degree III defects: histomorphometric outcomes

Degree III furcation involvements were surgically created at four first molars in each of three monkeys. Following 6 weeks of healing, full-thickness flaps were elevated. Following 24% EDTA gel conditioning, the defects were treated with one of the following: (1) enamel matrix proteins (EMD), (2) guided tissue regeneration (GTR) or (3) a combination EMD and GTR. The control defects did not receive any treatment. After 5 months of healing, the animals were sacrificed. Three 8 μm thick histological central sections, 100 μm apart, were used for histomorphometric analysis in six zones of each tooth either within the furcation area or on the pristine external surface of the root. In all specimens, new cementum with inserting collagen fibres was formed. Following GTR or GTR + EMD, cementum was formed up to and including the furcation fornix indicating complete regeneration on the defect periphery. Periodontal ligament fibres were less in all four modalities compared to pristine tissues. In the teeth treated with GTR and GTR + EMD a higher volume of bone and periodontal ligament tissues was observed compared to EMD. After 5 months of healing, regenerated tissues presented quantitative differences from the pristine tissues. In the two modalities where GTR alone or combined with EMD was used, the regenerated tissues differed in quantity from the EMD-treated sites.

Desmoglein-1 is a minor autoantigen in dogs with pemphigus foliaceus

The majority of human patients with pemphigus foliaceus (PF) have circulating IgG autoantibodies that target conformational epitopes on the desmosomal cadherin desmoglein-1 (dsg1). Limited studies using immunoblot techniques suggested that the principal autoantigen in dogs with PF might also be dsg1. It was the objective of this study to test this hypothesis. A comprehensive survey of canine PF sera was conducted using a novel screening strategy that detects conformational epitopes. This method consists of the ectopic expression of canine dsg1 at the surface of human 293T epithelial kidney cells and their live screening, i.e. prior to fixation. Out of seven control human PF sera that bound to canine epidermis, three (57%) contained IgG autoantibodies that recognized ectopically expressed canine dsg1 with a membrane and punctate pattern. Out of 83 canine PF sera only five (6%) contained IgG that recognized canine dsg1. Consistent with findings for human PF sera obtained in this study, autoantibody binding was conformation- and glycosylation-dependent as demonstrated by calcium chelation with EDTA and tunicamycin or wheat germ agglutinin treatment, respectively. In conclusion, these studies establish canine dsg1 as a minor autoantigen for canine PF. Antigenic epitopes appear to be conformation- and glycosylation-dependent.

Extrinsic causes of erosion. Oral hygiene products and acidic medicines

Acidic or EDTA-containing oral hygiene products and acidic medicines have the potential to soften dental hard tissues. The low pH of oral care products increases the chemical stability of some fluoride compounds, favors the incorporation of fluoride ions in the lattice of hydroxyapatite and the precipitation of calcium fluoride on the tooth surface. This layer has some protective effect against an erosive attack. However, when the pH is too low or when no fluoride is present these protecting effects are replaced by direct softening of the tooth surface. Xerostomia or oral dryness can occur as a consequence of medication such as tranquilizers, anti-histamines, anti-emetics and anti-parkinsonian medicaments or of salivary gland dysfunction e.g. due to radiotherapy of the oral cavity and the head and neck region. Above all, these patients should be aware of the potential demineralization effects of oral hygiene products with low pH and high titratable acids. Acetyl salicylic acid taken regularly in the form of multiple chewable tablets or in the form of headache powder as well chewing hydrochloric acids tablets for treatment of stomach disorders can cause erosion. There is most probably no direct association between asthmatic drugs and erosion on the population level. Consumers, patients and health professionals should be aware of the potential of tooth damage not only by oral hygiene products and salivary substitutes but also by chewable and effervescent tablets. Additionally, it can be assumed that patients suffering from xerostomia should be aware of the potential effects of oral hygiene products with low pH and high titratable acids.