Genetic and biochemical characterization of thermophilic ß-cyclodextrin glucanotransferase from Gracilibacillus alcaliphilus SK51.001.

BACKGROUND: Gracilibacillus alcaliphilus SK51.001, a strain that produces ß-CGTase (ß-cyclodextrin glucanotransferase) (EC 2.4.1.19), was screened and isolated from Sudanese soil. The objective of this study was to sequence and characterize the ß-CGTase gene from G. alcaliphilus SK51.001. RESULTS: According to 16S rRNA analysis of the strain and its morphological shape, it was identified as G. alcaliphilus. The ß-CGTase gene was successfully cloned, sequenced, and expressed in Escherichia coli BL21. This gene showed 706 amino acid residues including 33 amino acids as a signal peptide. The active site residues of G. alcaliphilus SK51.001CGTase were described using enzyme modeling and docking with the products. The estimated molecular mass of G. alcaliphilus SK51.001CGTase was approximately 74 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the evaluation of the gel filtration showed approximately 85 kDa, which means G. alcaliphilus SK51.001CGTase is a monomer. The optimum temperature and pH of G. alcaliphilus SK51.001CGTase were 60 °C and 7.0 respectively. Gracilibacillus alcaliphilus SK51.001CGTase was comparatively stable at a pH levels between 6.0 and 9.0 and temperatures of 30-50 °C. The activity of G. alcaliphilus SK51.001CGTase was increased by Ni2+ , and Co2+ but inhibited by Al3+ and Fe3+ . The kinetic parameters of Km and Vmax were 2068.52 µg mL-1 and 0.13 µmol mL-1  min-1 , respectively. CONCLUSION: Gracilibacillus alcaliphilus SK51.001CGTase could hydrolyze soluble starch into α-, ß-, and γ-cyclodextrin in a ratio of 2: 83: 15% respectively. This high ratio production of ß-CD could allow the enzyme to be used in ß-CD production. © 2020 Society of Chemical Industry.

Ruminal fermentation and microbial community differently influenced by four typical subtropical forages in vitro.

The present study evaluated the effects of 4 typical subtropical forages on ruminal microbial community composition to formulate a better diet for buffalo. Corn straw silage, elephant grass, cassava residues and sugarcane tail silage were used as substrates for in vitro fermentation. Eight replicates were set up for every substrate, and fermentation was carried out in a 100-mL glass syringe, using buffalo rumen inoculum. Every replicate was anaerobically dispensed with 10 mL of rumen inoculum, 20 mL of McDougall's buffer and 200 mg of dried substrate, and placed in a water bath at 39 °C. Gas production was recorded at 0, 2, 6, 12, 24, 36, 48 and 72 h of incubation. After 24 h, fermentation was ceased for 4 replicates and samples were collected. Volatile fatty acids (VFA) concentrations were measured using gas chromatography. Microbial populations were quantified using quantitative real-time PCR (qRT-PCR), and microbial community was analyzed using high throughput sequencing technology. The results showed, cassava residues as substrate had the highest gas production, acetate, propionate and total VFA concentrations (P < 0.05), and corn straw silage had the lowest acetate:propionate ratio (P < 0.05). The lowest numbers of fungi, Ruminococcus albus and Fibrobacter succinogenes, and the highest number of protozoa were observed with cassava residues (P < 0.05). The least abundances of bacterial phyla Firmicutes, Bacteroidetes and genus Prevotella, and substantially higher abundance of phylum proteobacteria (56%) and genus Succinivibrio (52%) were observed with cassava residues. The most abundances of Methanobrevibacter gottschalkii and Entodinium were observed with cassava residues. Spearman's correlations analysis showed, Succinivibrio had strong positive correlations with propionate, butyrate, Metadinium and M. gottschalkii, indicating fermentation products were related to microbial community. In conclusion, incubation with cassava residues resulted in lower number of fiber degrading microbes but higher protozoal population because of its low fiber contents. The microbial community was highly altered by in vitro incubation with cassava residues, whereas remained similar for the other 3 high fiber containing substrates.

Does body mass index impact the outcomes of tubeless percutaneous nephrolithotomy?

OBJECTIVE: To evaluate whether body mass index (BMI) has an impact on the outcomes of tubeless percutaneous nephrolithotomy (PCNL). PATIENTS AND METHODS: We retrospectively reviewed patients who underwent tubeless PCNL at our institution from 2006 to 2011. Specifically, stone-free rates, complications, and hospital length of stay (LOS) were assessed. Patients were divided into four groups based on BMI: <25, 25-29.9, 30-34.9 and ≥35 kg/m(2) . Baseline characteristics and outcomes were compared between BMI groups. Multivariable logistic regressions were used to evaluate the independent contribution of BMI as a predictor of outcomes. RESULTS: We identified 268 patients who fulfilled study requirements. The overall stone-free and complication rates were 52.5% and 19.0%, respectively. Minor and severe complication comprised 10.4% and 8.6%, respectively. Univariate and multivariable analyses showed no association between BMI and stone-free or complication rates. However, patients with a normal BMI had significantly higher transfusion rates (P = 0.005), and were significantly more likely to have a prolonged LOS (≥2 days), when compared with an overweight BMI (P = 0.032) CONCLUSIONS: BMI did not impact the stone-free, or complication rates of tubeless PCNL. Normal BMI was found to be a risk factor for prolonged LOS, which may be due to an increase in clinically significant bleeding in this patient population. Tubeless PCNL appears to be a safe and effective procedure for the treatment of complex renal calculi, independent of BMI.

Most infants with dilating vesicoureteral reflux can be treated nonoperatively.

PURPOSE: Dilating vesicoureteral reflux provokes concern for physicians and parents that often leads to corrective surgery in young children. Since there are limited data describing the natural history of dilating vesicoureteral reflux in infants, we identified factors predictive of resolution/improvement in infants initially treated nonoperatively. MATERIALS AND METHODS: We reviewed the medical records of 90 infants 6 months old or younger from 2004 to 2010 who were referred for prenatal hydronephrosis or initial febrile urinary tract infection and found to have dilating vesicoureteral reflux (grade 3 or greater). Variables of interest included presentation, dimercapto-succinic acid results, sex, breakthrough febrile urinary tract infections, reflux grade and bilateral reflux. Cox regression analysis was performed to determine predictors of spontaneous resolution and/or improvement to reflux grade less than 3 as well as predictors of surgical intervention. RESULTS: Included in final analysis were 80 infants (113 renal units). Of the patients 51 (64%) experienced spontaneous resolution/improvement with a mean followup of 29 months before resolution, discharge home and/or end of followup. Only 20 patients (25%) underwent surgery. Cox regression analysis revealed that a normal initial dimercapto-succinic acid scan, initial reflux grade less than 5 and absent breakthrough febrile urinary tract infections were predictive of reflux resolution/improvement (p <0.05). Dimercapto-succinic acid scan abnormalities, prenatal hydronephrosis and breakthrough febrile urinary tract infections were significant predictors of surgery (p <0.05). CONCLUSIONS: Dilating vesicoureteral reflux in infancy often resolves/improves spontaneously. Therefore, surgery should be directed toward patients unlikely to experience resolution, ie those with an abnormal initial dimercapto-succinic acid scan, grade 5 vesicoureteral reflux and breakthrough febrile urinary tract infections.

Increasing dwell time of mitomycin C in the upper tract with a reverse thermosensitive polymer.

UNLABELLED: Abstract Background and Purpose: Topical chemotherapy for urothelial cancer is dependent on adequate contact time of the chemotherapeutic agent with the urothelium. To date, there has not been a reliable method of maintaining this contact for renal or ureteral urothelial carcinoma. We evaluated the safety and feasibility of using a reverse thermosensitive polymer to improve dwell times of mitomycin C (MMC) in the upper tract. MATERIALS AND METHODS: Using a porcine model, four animals were treated ureteroscopically with both upper urinary tracts receiving MMC mixed with iodinated contrast. One additional animal received MMC percutaneously. The treatment side had ureteral outflow blocked with a reverse thermosensitive polymer plug. MMC dwell time was monitored fluoroscopically and intrarenal pressures measured. Two animals were euthanized immediately, and three animals were euthanized 5 days afterward. RESULTS: In control kidneys, drainage occurred at a mean of 5.3±0.58 minutes. Intrarenal pressures stayed fairly stable: 9.7±14.0 cm H20. In treatment kidneys, dwell time was extended to 60 minutes, when the polymer was washed out. Intrarenal pressures in the treatment kidneys peaked at 75.0±14.7 cm H20 and reached steady state at 60 cm H20. Pressures normalized after washout of the polymer with cool saline. Average washout time was 11.8±9.6 minutes. No histopathologic differences were seen between the control and treatment kidneys, or with immediate compared with delayed euthanasia. CONCLUSIONS: A reverse thermosensitive polymer can retain MMC in the upper urinary tract and appears to be safe from our examination of intrarenal pressures and histopathology. This technique may improve the efficacy of topical chemotherapy in the management of upper tract urothelial carcinoma.

Obesity triples the radiation dose of stone protocol computerized tomography.

PURPOSE: Patients with recurrent nephrolithiasis are often evaluated and followed with computerized tomography. Obesity is a risk factor for nephrolithiasis. We evaluated the radiation dose of computerized tomography in obese and nonobese adults. MATERIALS AND METHODS: We scanned a validated, anthropomorphic male phantom according to our institutional renal stone evaluation protocol. The obese model consisted of the phantom wrapped in 2 Custom Fat Layers (CIRS, Norfolk, Virginia), which have been verified to have the same radiographic tissue density as fat. High sensitivity metal oxide semiconductor field effect transistor dosimeters were placed at 20 organ locations in the phantoms to measure organ specific radiation doses. The nonobese and obese models have an approximate body mass index of 24 and 30 kg/m(2), respectively. Three runs of renal stone protocol computerized tomography were performed on each phantom under automatic tube current modulation. Organ specific absorbed doses were measured and effective doses were calculated. RESULTS: The bone marrow of each model received the highest dose and the skin received the second highest dose. The mean ± SD effective dose for the nonobese and obese models was 3.04 ± 0.34 and 10.22 ± 0.50 mSv, respectively (p <0.0001). CONCLUSIONS: The effective dose of stone protocol computerized tomography in obese patients is more than threefold higher than the dose in nonobese patients using automatic tube current modulation. The implication of this finding extends beyond the urological stone population and adds to our understanding of radiation exposure from medical imaging.

In-vitro assessment of a new portable ballistic lithotripter with percutaneous and ureteroscopic models.

BACKGROUND AND PURPOSE: The EMS Swiss LithoBreaker is a new, portable, electrokinetic lithotripter. We compared its tip velocity and displacement characteristics with a handheld, pneumatic lithotripter LMA StoneBreaker.™ We also evaluated fragmentation efficiency using in vitro models of percutaneous and ureteroscopic stone fragmentation. MATERIALS AND METHODS: Displacement and velocity profiles were measured for 1-mm and 2-mm probes using a laser beam aimed at a photo detector. For the percutaneous model, 2-mm probes fragmented 10-mm spherical BegoStone phantoms until the fragments passed through a 4-mm mesh sieve. The ureteroscopic model used 1-mm probes and compared the pneumatic and electrokinetic devices to a 200-µm holmium laser fiber. Cylindrical (4-mm diameter, 4-mm length) BegoStone phantoms were placed into silicone tubing to simulate the ureter; fragmented stones passed through a narrowing in the tubing. RESULTS: For both 1-mm and 2-mm probes, the electrokinetic device had significantly higher tip displacement and slower tip velocity, P<0.01. In the percutaneous model, the electrokinetic device needed an average of 484 impulses over 430 seconds to fragment one BegoStone, while the pneumatic device needed 29 impulses over 122 seconds to fragment one stone. Both clearance times and number of impulses needed for percutaneous stone clearance were significantly different at P<0.01. Ureteroscopically, the mean clearance time was 97 seconds for the electrokinetic lithotripter, 145 seconds for the pneumatic lithotripter, and 304 seconds for the laser. Comparing the pneumatic device with the electrokinetic device ureteroscopically, there was no significant difference in clearance time, P=0.55. Both the pneumatic and electrokinetic lithotripters, however, demonstrated decreased clearance times compared with the laser, P=0.027. CONCLUSIONS: The portable electrokinetic lithotripter may be better suited for ureteroscopy instead of percutaneous nephrolithotomy. It appears to be comparable to the portable pneumatic device in the ureter. Further clinical studies are needed to confirm these findings in vivo.

Optical performance comparison of deflectable laparoscopes for laparoendoscopic single-site surgery.

BACKGROUND AND PURPOSE: Flexible working angles and fine optical visualization are major requisite factors in performing laparoendoscopic single-site (LESS) urologic procedures. Multiple mechanical design approaches have been used to develop deflectable laparoscopes for LESS procedures. We compared the optical characteristics of three such devices using a bench top approach to simulate LESS in straight and deflected positions. MATERIALS AND METHODS: A 10-mm fixed-rod rotating lens device (Storz EndoCameleon) and two 5-mm articulating devices (Olympus EndoEye and Stryker IdealEye) were compared using standard industry testing protocols for image resolution (United States Air Force-1951 test target), distortion (multifrequency grid distortion target), and color reproducibility (Gretag Macbeth color checker). RESULTS: The 10-mm fixed-rod rotating lens system demonstrated the highest image resolution (5.04 line pairs/mm), but also the highest distortion (22.8%). Among the 5-mm flexible articulating laparoscopes, resolution was superior with the Olympus EndoEye (4.00 line pairs/mm) compared with the Stryker IdealEye (3.17 line pairs/mm). Distortion (7.0%) and color reproduction (1.18) were superior with the IdealEye vs the EndoEye (18.8 %, 1.27). Laparoscope deflection resulted in attenuation of resolution by 11% with both articulating models, but not with the fixed rod system. CONCLUSIONS: Definition of these optical characteristics may inform further development and selection of laparoscopic systems optimized for LESS surgery. A narrow but flexible camera can be crucial in the limited working space available during these procedures. Further investigation is warranted to determine if these objective findings translate into improved surgeon performance.