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Clostridium fermentations have been developed for producing butanol and other value-added chemicals, but their development is constrained by some limitations, such as relatively high substrate cost and the need to maintain an anaerobic condition. Recently, co-culture is emerging as a popular way to address these limitations by introducing a partner strain with Clostridium. Generally speaking, the co-culture strategy enables the use of a cheaper substrate, maintains the growth of Clostridium without any anaerobic treatment, improves product yields, and/or widens the product spectrum. Herein, we review recent developments of co-culture strategies involving Clostridium species according to their partner stains' functions with representative examples. We also discuss research challenges that need to be addressed for the future development of Clostridium co-cultures. Clostridium co-culture has emerged as a solution to some problems faced by Clostridium monoculture. Co-culturing Clostridium with cellulolytic strains or acetogens enables the production of butanol from cheaper substrates such as lignocellulosic biomass and syngas. Aerotolerance is possible in anaerobic Clostridium fermentation in co-cultures with oxygen-consuming microbes. Co-culturing solventogenic Clostridium with acetogens can improve the production of butanol and its derivatives by increasing carbon recovery and relieving solvent toxicity. Other products, such as H 2 or fatty acids, can be efficiently produced by the co-culture of Clostridium and other H 2 -producing strains (such as PNS bacteria), or chain-elongating strains respectively. [ABSTRACT FROM AUTHOR]

Abnormal protease activities are associated with cancers, vascular diseases and Alzheimer diseases. Therefore, detection of protease activities has become increasingly important in recent years. Herein, we report a semi-quantitative, liquid crystal (LC)-based protease assay for naked-eye detection of protease activity. In this assay, casein molecules are cleaved by proteases into small peptide fragments, which can be quantified either by using Lowry’s method or using LC. In the latter, peptide fragments adsorb on a solid surface and disrupt LC to produce a bright spot for naked-eye detection. The bright spot is observed only when the surface-adsorbed peptide density exceeds a critical value. In the assay, a major challenge is how to separate undigested casein and remaining protease from peptide fragments to prevent their interferences with LC. To overcome this issue, trichloroacetic acid (TCA) is added to precipitate casein and proteases. By using the assay, we are able to detect 10 ng/mL of protease (activity 7.23 U/mg protease, R 2  = 0.991) or 6.5 μg/mL of peptide fragments. Finally, a continuous protease assay is developed to minimize manual sampling and reduce errors in kinetic studies. [ABSTRACT FROM AUTHOR]

Cascade reactions involved unstable intermediates are often encountered in biological systems. In this study, we developed combined cross-linked enzyme aggregates (combi-CLEA) to catalyze a cascade reaction which involves unstable hydrogen peroxide as an intermediate. The combi-CLEA contains two enzymes̶ glucose oxidase (GOx) and horseradish peroxidase (HRP) which are cross-linked together as solid aggregates. The first enzyme GOx catalyzes the oxidation of glucose and produces hydrogen peroxide, which is used by the second enzyme HRP to oxidize 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS). The apparent reaction rate of the cascade reaction reaches 10.5 ± 0.5 μM/min when the enzyme ratio is 150:1 (GOx:HRP). Interestingly, even in the presence of catalase, an enzyme that quickly decomposes hydrogen peroxide, the reaction rate only decreases by 18.7% to 8.3 ± 0.3 μM/min. This result suggests that the intermediate hydrogen peroxide is not decomposed by catalase due to a short diffusion distance between GOx and HRP in the combi-CLEA. Scanning electron microscopy images suggest that combi-CLEA particles are hollow spheres and have an average diameter around 250 nm. Because of their size, combi-CLEA particles can be entrapped inside a nylon membrane for detecting glucose by using the cascade reaction. [ABSTRACT FROM AUTHOR]

Butanol and butyric acid produced from acetone-butanol-ethanol (ABE) fermentation can be used to produce butyl butyrate, an important fragrance ester. However, low levels of butanol and butyric acid need to be purified from culture media first with energy-intensive distillation processes. In this study, a triphasic (organic/aqueous/fluorous) system is developed to esterify butanol and butyric acid in spent culture media into butyl butyrate directly without purification. The produced butyl butyrate forms a distinct organic phase floating on top and can then be separated easily. In a model system containing 37.1 g/L of butanol and 44.1 g/L of butyric acid, 57% of the butanol is converted to butyl butyrate after 8 h of esterification. With multiple cycles of esterification and product removal, butanol conversion can be further increased to 86%. When spent culture medium containing 7.12 g/L of butanol and 4.81 g/L of butyric acid is used for esterification, 38% of butanol (0.36 mmol) is consumed and 0.33 mmol of butyl butyrate is produced. However, when ABE fermentation and esterification are carried out simultaneously, only 0.042 mmol of butyl butyrate is produced, probably due to the incompatible pH requirements for cell growth (pH 5–7) and esterification (pH 2–3). [ABSTRACT FROM AUTHOR]

Polyethylene glycol (PEG) hydrogel is permeable to biomolecules, but its permeability depends on the molecular weight of monomers and the concentration of monomer solutions. In this study, we show that PEG hydrogel made from 20% to 30% of PEG700 monomer is permeable to amino acids yet impermeable to oligopeptides. Because of its unique permeability, the gel can be used to detect protease by separating amino acids from oligopeptides when proteases cleave the oligopeptides and release amino acids. Based on this principle, an UV crosslinked gel array is fabricated on a chip for simultaneous detection of protease in up to 40 samples with only 1 µl of volume required for each sample. As a proof of concept, the on-chip protease assays are used to detect trypsin in buffer and serum. The detection limits are 1.2 nM in buffer and 17.7 nM in serum, which are comparable to conventional protease assays. Moreover, because only 1 µl of liquid is required, as little as 1.2 fmol of trypsin can be detected by using the on-chip assay. The protease assay also shows good specificity for trypsin and chymotrypsin. The gel array chip could be a useful miniaturized platform for high-throughput detection of different proteases and screening of their inhibitors. [ABSTRACT FROM AUTHOR]

Esterification of organic acids and alcohols in aqueous media is very inefficient due to thermodynamic constraints. However, fermentation processes used to produce organic acids and alcohols are often conducted in aqueous media. To produce esters in aqueous media, biphasic alginate beads with immobilized lipase are developed for in situ esterification of butanol and butyric acid. The biphasic beads contain a solid matrix of calcium alginate and hexadecane together with 5 mg/mL of lipase as the biocatalyst. Hexadecane in the biphasic beads serves as an organic phase to facilitate the esterification reaction. Under optimized conditions, the beads are able to catalyze the production of 0.16 mmol of butyl butyrate from 0.5 mmol of butyric acid and 1.5 mmol of butanol. In contrast, when monophasic beads (without hexadecane) are used, only trace amount of butyl butyrate is produced. One main application of biphasic beads is in simultaneous fermentation and esterification (SFE) because the organic phase inside the beads is very stable and does not leach out into the culture medium. SFE is successfully conducted with an esterification yield of 6.32% using biphasic beads containing iso-octane even though the solvent is proven toxic to the butanol-producing Clostridium spp. [ABSTRACT FROM AUTHOR]

Clostridium beijerinckii G117 produces hydrogen (∼4400 mL/L) from 15 g/L of native crude glycerol (CG). While increasing the native-CG to 20–80 g/L, strain G117 rejects 34–90% of CG and fails to support a relative enhancement in hydrogen production. Subsequently, to enhance the native-CG assimilation, and hydrogen production from strain G117, various agro-residual hydrolysates (ARH), and several metal-ions have individually tested as media-supplements. Among tested, rice bran hydrolysate (RBH) and ferrous chloride (Fe2+) are selected as media-supplements. By employing RSM-based central composite design, the medium composed of 37.63 g/L CG, 3.65 g/L RBH and 1.11 mM Fe2+ is optimized which results a favorable initial redox potential (−191.0 ± 1.5 mV), higher cellular growth (6.73 ± 0.12 OD 600), and 17350 ± 170 mL/L of hydrogen production from strain G117. The analysis of total-mass and electron balances suggest that this bioprocess is remarkably efficient (82–99%), and records the highest yield of hydrogen (2.27 ± 0.02 mol/mol glycerol) ever reported in literature. [Display omitted] • Clostridium beijerinckii G117 produces hydrogen from <15 g/L of crude glycerol (CG). • While raising CG to 20–80 g/L in the media, strain G117 rejects 34–90% of CG supplied. • Adding agro-residual hydrolysates/metal-ions alter CG-utilization and hydrogen yield. • Media of CG with rice bran hydrolysate as co-substrate and Fe2+ is optimized (OpM). • OpM supports G117 to produce 17350 mL/L hydrogen with yield of 2.27 mol H2 /mol Glycerol. [ABSTRACT FROM AUTHOR]

We reported an oligopeptide immobilization strategy for improving stability and sensitivity of liquid-crystal (LC) based protease assay. In this strategy, oligopeptides are immobilized by using N -terminal cysteine or free amine groups on lysine residues to react with surface aldehyde groups and form multiple anchoring points. Our results show that the number of anchoring points is an important factor for improving stability of the immobilized oligopeptides. Oligopeptides with multiple anchoring points are very stable on the surface. They can only be cleaved from the surface by using proteases such as trypsin or α-chymotrypsin. This phenomenon provides a principle for developing an assay for the detection of proteases. On the other hand, oligopeptides with a single anchoring point cannot form stable linkage on the surface, and they can be easily washed off during rinsing steps. By immobilizing oligopeptides with different surface densities, concentrations of trypsin and α-chymotrypsin can be estimated simply by counting the number of bright LC spots. The higher the concentrations of trypsin and α-chymotrypsin, the less LC spots can be observed. In addition, the limit of detection (LOD) of the assay can be lowered by allowing a very large oligopeptide fragment to desorb from the surface after the cleavage. This task can be accomplished by immobilizing an oligopeptide with all anchoring points located at both ends while the cleavage sites are located near the anchoring points. Based on the principle, optimized LODs for trypsin and α-chymotrypsin can reach 1 and 0.1 ng/mL, respectively. [ABSTRACT FROM AUTHOR]

Abstract: We report a biosensor for detecting organophosphates (OPs) by using liquid crystal (LC). The mechanism of the biosensor is based on detection of minute pH changes during the enzymatic hydrolysis of OPs. To hydrolyze OPs, paraoxonase 1 (PON1) is immobilized on a copper grid which also hosts pH-sensitive LC, 4-cyano-4′-pentylbiphenyl (5CB) doped with 0.3% of 4′-pentyl-biphenyl-4-carboxylic acid (PBA). Proximity of enzyme and LC ensures that H+ can be detected by the pH-sensitive LC before it is neutralized by buffer. Thus, the presence of OPs can be reported as color change in LC when they undergo enzymatic hydrolysis. For paraoxon detection, we can reach a limit of detection (LOD) of 1μM by using the naked eye. This type of LC-based biosensor also shows high specificity and does not respond to imidacloprid and ampicillin. [Copyright &y& Elsevier]

Abstract: A liquid crystal (LC)-based optical sensor has been developed to detect vaporous butylamine in air. This LC sensor doped with lauric aldehyde (LA) shows fast and distinct bright-to-dark optical response to butylamine vapor. For example, when the LA doping concentration is 0.1wt%, the LC shows a rapid bright-to-dark optical response within 2min after it is exposed to 10ppmv of butylamine. This optical response is attributed to an orientational transition of LC triggered by a reaction between LA and butylamine. This LC sensor also exhibits reversibility after the sensor is exposed to open air. In addition to primary amines (such as butylamine and octylamine), this LC-based sensor responds to secondary amines (such as diisopropylamine), but the detection limit is 200ppmv, which is much higher than butylamine. [Copyright &y& Elsevier]

• A liquid crystal-based protease inhibition assay is developed. • This assay is used to assess inhibition efficiency of a protease inhibitor pefabloc. • IC 50 value of pefabloc can be detected by using the color of liquid crystal. • The assay provides detailed information about the transition period from reversible to irreversible inhibition. • Pefabloc inhibits protease activity irreversibly if the incubation time is more than 60 min. Protease inhibitors are essential drug molecules which can be used for treatments of protease-related diseases. Herein, we report an LC-based protease inhibition assay built inside a millifluidic device. This assay can be used to study the inhibition efficiency and reversibility of pefabloc (a serine protease inhibitor) against proteases. LC is employed for the detection of peptide fragments produced from protease activity inside the millifluidic device. LC gives a bright spot when the amount of peptide fragment exceeds a minimum value. By using this assay, we find that IC 50 value of pefabloc for the immobilized protease is 0.45 mg/mL, which is lower than the IC 50 value of pefabloc (0.90 mg/mL) obtained in a homogeneous assay. Moreover, proteases can be immobilized on the millifluidic device to build a heterogeneous protease assay. In this assay, pefabloc blocks the immobilized protease irreversibly after 60 min, which is longer than that in a homogenous protease assay. This reversibility study provides useful information about the transition period of pefabloc from reversible to irreversible inhibition. This protease inhibition assay is potentially useful for high throughput screening of unknown proteases and their inhibitor in a small sample volume. Moreover, this method can be used for dosage test of novel drug molecules which are protease inhibitors. [ABSTRACT FROM AUTHOR]

Butanol toxicity, oxygen sensitivity, and high substrate cost limit wider applications of the traditional Clostridium acetone-butanol-ethanol (ABE) fermentation. In this study, a wild-type Clostridium beijerinckii strain was partnered with an engineered Escherichia coli strain to improve the ABE fermentation through a co-culture. Butanol and butyrate produced by C. beijerinckii were converted into butyl butyrate – a product that can be in situ removed to reduce product toxicity – by expressing a CoA transferase and an alcohol acyltransferase in E. coli. When integrated with a pretreatment technology and using commercial cellulase, the co-culture produced 1280 mg/L butyl butyrate from rice straw in bioreactors, without the need of maintaining a strict anaerobic condition. As a process harnessing both the genetic tractability of E. coli and the superior acid- and alcohol-producing ability of Clostridium , this co-culture fermentation will be useful in producing valuable esters from low-cost waste streams. [Display omitted] • E. coli was engineered to express a CoA transferase and an alcohol acyltransferase. • Butyl butyrate was produced by the co-culture from lignocellulosic biomass. • Aerobic co-culture of E. coli and Clostridium was achieved. [ABSTRACT FROM AUTHOR]

Xylooligosaccharides (XOS) are emerging prebiotics which can be produced from lignocellulosic biomass including agro-residues and hardwood. In this study, we report the production of XOS from thermal-alkali pretreated hardwood such as mahogany and mango by using a purified xylanase from Clostridium strain BOH3. In the first approach, pure xylan is extracted from mahogany and mango hardwood and then the pure xylan is hydrolyzed by using the xylanase. In this case, 572 and 504 mg XOS/g pure xylan were obtained from mahogany and mango woods, respectively. In the second approach, the same xylanase is employed to hydrolyze sawdust of hardwood after different types of pretreatments. After a thermal (121 °C for 15 min) pretreatment under a mild alkaline (0.05N NaOH) condition, the pretreated mahogany and mango sawdust can be utilized directly to produce 89.5 and 67.6 mg XOS/g pretreated sawdust, respectively. XOS produced from the pretreated sawdust show strong prebiotic effects on Bifidobacteria and Lactobacilli . This report shows the possibility of producing XOS from pretreated woody wastes without using pure xylan as a substrate. [ABSTRACT FROM AUTHOR]

Clostridia can produce hydrogen (a renewable-biofuel) from crude glycerol (CG). Reportedly, the indigenous CG-impurities (eg.methanol, soap and salt) interfere with clostridial-process, and reduces hydrogen production. Thus, it is important to develop a robust bioprocess to directly utilize native-CG for hydrogen production. In this study, among four Clostridia tested, Clostridium beijerinckii G117 is selected based on its high hydrogen (>290 mL/g) production from CG. Furthermore, optimization of process-parameters enhances hydrogen production from strain G117 by ∼5 fold from 1195 ± 45 to 5893 ± 25 mL/L with 2.16 mol/mol yield. The order of impact of process-parameters (optimum value in parenthesis) on hydrogen production is: CG concentration (12.5 g/L)>inoculum size (10%v/v)>inoculum age (12 h)>temperature (39 °C)>initial medium-pH (6.4)>L-cysteine (1 g/L)>operational volume (30 mL)>agitation rate (150 rpm). Notably, this optimized bioprocess records >90% efficiency by determining total mass and electron balances. Interestingly, this optimized bioprocess retains adequate robustness to yield 1.94–2.02 mol/mol hydrogen with elevated CG-impurities including alcohols/salts/surfactants. [Display omitted] • Clostridium beijerinckii G117 produces >290 mL hydrogen per gram crude glycerol (CG). • Optimization of process with strain G117 enhances hydrogen production to >520 mL/g CG. • Along with hydrogen, strain G117 produces 0.7 g/L acetic, and 2.71 g/L butyric acids. • Strain G117 is resistant to many CG-impurities including alcohols/salts/surfactants. • It renders an efficient (>90%) and robust-bioprocess to yield ∼2 mol H2 /mol Glycerol. [ABSTRACT FROM AUTHOR]

In this study, a fermentation process for production of butanol and butyl-butyrate by using Clostridium sp. strain BOH3 is developed. This strain is able to produce butyric acid and butanol when it ferments 60 g/L xylose. Meanwhile, it also excreted indigenous lipases (induced by olive oil) which naturally convert butyric acid and butanol into 1.2 g/L of butyl-butyrate. When Bio-OSR was used as both an inducer for lipase and extractant for butyl-butyrate, the butyl-butyrate concentration can reach 6.3 g/L. To further increase the yield, additional lipases and butyric acid are added to the fermentation system. Moreover, kerosene was used as an extractant to remove butyl-butyrate in situ . When all strategies are combined, 22.4 g/L butyl-butyrate can be produced in a fed-batch reactor spiked with 70 g/L xylose and 7.9 g/L butyric acid, which is 4.5-fold of that in a similar system (5 g/L) with hexadecane as the extractant. [ABSTRACT FROM AUTHOR]

Solventogenic Clostridium strains usually do not ferment lignocellulosic biomass directly due to insufficient expression of hydrolyzing enzymes. In this study, we show that a solventogenic Clostridium strain BOH3 is able to produce both butanol and hydrogen when it ferments mixtures of agricultural residues and oil cakes. In particular, fermentation of mixed rice bran and sesame oil cake gives the highest butanol and hydrogen concentrations. In an optimized agro-residual medium (OAM) containing 94.5 g/l of rice bran and 36.7 g/l of sesame oil cake, BOH3 produces 13.50 ± 0.12 g/l of butanol and 4.41 ± 0.04 l/l of hydrogen after 7 days. Because BOH3 excretes hydrolyzing enzymes such as cellulase (0.52 ± 0.08 U/ml), xylanase (4.10 ± 0.05 U/ml) and amylase (2.05 ± 0.12 U/ml) during fermentation, BOH3 is able to perform fermentation and saccharification simultaneously when rice bran and sesame oil cakes are used as substrates. This is the first report showing that direct fermentation of agricultural residues by using solventogenic Clostridia is possible. [ABSTRACT FROM AUTHOR]

In this study, a novel carbon fiber brush (CFB) electrode was designed using carbon fiber filaments and conductive metals. It was used as the cathode to construct an efficient coupled electro-Fenton and electrocoagulation (EF-EC) process for tetracycline (TC) treatment. An optimal 97.9% removal rate of 10 mg L−1 TC was achieved within 20 min. The coupled process is less pH-dependent and more effective in treating TC compared to the traditional individual electro-Fenton (EF) or electrocoagulation (EC) process, achieving efficient TC removal under neutral pH conditions. The removal rate of 10 mg L−1 TC consistently remained above 92% at 20 min after ten cycle experiments using the same electrodes in a Fe-CFB system (92.7–97.9%), indicating excellent reusability and stability of the CFB cathode. Mechanism analysis showed both EF and EC processes were involved in the system. Radicals (such as •OH and SO 4 −•) generated by EF contributed to the degradation of TC, yielding nine intermediates. Coagulants (such as Fe(OH) 3) generated by EC contributed to the removal of TC. Toxicity prediction results indicated that over half of the nine intermediates exhibited lower biotoxicity compared to TC. This study provides a feasible alternative cathode for the efficient treatment of TC using EF-EC process. [Display omitted] • An efficient EF-EC process was constructed using a novel CFB cathode and a Fe anode. • The EF-EC process achieved synchronized degradation and removal of TC. • •OH and SO 4 −• contributed to TC degradation and Fe(OH) 3 contributed to TC removal. • The EF-EC process achieved an optimal TC removal rate of 97.9% within 20 min. • Toxicity predictions from TEST indicated Fe-CFB system reduced the biotoxicity of TC. [ABSTRACT FROM AUTHOR]

Enzymatic hydrolysis of cellulose is often conducted in batch processes in which hydrolytic products tend to inhibit enzyme activity. In this study, we report a method for continuous hydrolysis of carboxymethyl cellulose (CMC) by using cross-linked cellulase aggregate (XCA) trapped inside a membrane. XCA particles prepared by using a millifluidic reactor have a uniform size distribution around 350 nm. Because of their large size, XCA particles in solutions can be filtered through a polyethersulfone membrane to collect 87.1 ± 0.9% of XCA particles. The membrane with impregnated XCA can be used as a catalyst for hydrolysis of CMC in a continuous mode. When the CMC concentration is 1.0 g/l and the flow rate is 2 μl/min, 53.9% of CMC is hydrolyzed to reducing sugars. The membrane with XCA is very stable under continuously flowing solutions. After 72 h of reaction, 97.5% of XCA remains inside the membrane. [ABSTRACT FROM AUTHOR]

Abstract: A colorimetric protease assay based on the cleavage of gold nanoparticles (AuNPs) aggregates cross-linked with oligopeptides is developed. In this assay, when oligopeptide YHPQMNPYTKAGGGC is added to a solution containing citrate-stabilized AuNPs, cysteine and lysine on the oligopeptide causes rapid aggregation of AuNPs, resulting in color change from red to blue. However, in the presence of a model protease trypsin, the oligopeptide is cleaved by trypsin into two fragments. As a result, AuNPs do not aggregate and no color change can be observed. By using this principle, trypsin can be detected with the naked eye at a concentration as low as 0.5nM. This assay can also be applied to detect trypsin inhibitors such as benzamidine hydrochloride. [Copyright &y& Elsevier]

Highlights: [•] Clostridium strain BOH3 is able to use xylan for its growth and ABE fermentation. [•] Optimized culture medium (OCM) enhances xylanase expression up to 1.6-fold. [•] In OCM, strain BOH3 can effectively utilize high concentration (30–50g/l) of xylan. [•] BOH3 produces >14g/l of butanol and >2.6l/l of hydrogen from xylan in OCM. [Copyright &y& Elsevier]

Abstract: We report an easily visualized liquid crystal (LC)-based system to study the biophysical interactions between protein-coated gold nanoparticles (AuNPs) and LC-supported cell membrane model. The model consists of mixed phospholipid/cholesterol monolayer self-assembled at aqueous–LC interface. Protein-coated AuNPs were found to disrupt the mixed phospholipid/cholesterol monolayer. As a result, orientational transitions of LCs were triggered and optical responses of LCs from dark to bright were observed. The mixed monolayers with higher cholesterol contents were found to be more susceptible to the disruption by protein-coated AuNPs, and hydrophobic interaction played a major role in the monolayer disruption. We also found that the time for non-specific binding of fibrinogen-coated AuNPs to the mixed phospholipid/cholesterol monolayer was similar to that of specific binding of neutravidin-coated AuNPs to the mixed phospholipid/biotin-capped phospholipid monolayer. Results obtained from this study may offer new understanding in the potential nanotoxicity pathway, where the biophysical interaction between nanomaterials and cell membrane is an important step. [Copyright &y& Elsevier]

Abstract: We report an easily visualized liquid crystal (LC)-based system to study the molecular interactions between protein-coated gold nanoparticles (AuNPs) and supported phospholipid monolayer self-assembled at the aqueous-LC interface. Protein-coated AuNPs were found to disrupt the phospholipid monolayer and resulted in the orientational transitions of LCs that support the phospholipid layer. The disruption of the phospholipid monolayer depends on the type of protein (albumin, neutravidin, and fibrinogen) adsorbing onto nanoparticles. Furthermore, our results suggest that hydrophobic interaction plays a major role in the disruption of the phospholipid layer by protein-coated AuNPs. Results obtained from this study may offer new understanding in the potential cytotoxicity of nanomaterials, where the interaction between nanoparticles and cell membrane is an important step. [Copyright &y& Elsevier]

Objective: To illustrate our technique to construct the Institute of Urology Peking University (IUPU) orthotopic ileal bladder and present our initial experience.Methods: From August 2017 to April 2018, 12 patients with bladder cancer underwent radical cystectomy (RC), pelvic lymph node dissection and extracorporeal construction of an IUPU neobladder (IUPUB) by an experienced surgeon. We present the demographic, clinicopathologic, perioperative, and follow-up data. We also describe our step-by-step surgical technique for the IUPUB in this article.Results: Laparoscopic RC with an extracorporeal IUPUB was successfully accomplished in 11 patients, and 1 patient was converted to open RC with an IUPUB. The median total operative time and median time spent suturing the pouch were 248 minutes and 23 minutes, respectively. The median estimated blood loss was 150 mL. The median time to recovery of bowel function (tolerance of a liquid diet) was 3½ days. The urinary catheter was removed on post-operative day 21 in 10 patients. The ureteral stents and stoma catheter were removed on day 7 after cystography. At a median followup of 7½ months, 2 patients had early complications (<30 days), and no major complications (grade ≥ 3) occurred. The follow-up outcomes were satisfactory. The limitations included the small sample size and short-term outcomes.Conclusion: Our technique of constructing the IUPUB is feasible and safe. The operative time and early complication rates are acceptable. [ABSTRACT FROM AUTHOR]