Cloning, Characterization of Pichia etchellsii β-Glucosidase II and Effect of Media Composition and Feeding Strategy on its Production in a Bioreactor

Saroj Mishra,Benu Sethi,Monika Jain,Manish Chowdhary,Yogesh Soni,Yukti Bhatia,Vikram Sahai 한국생물공학회 2002 Biotechnology and Bioprocess Engineering Vol.7 No.1

The cloning and expression of β-glucosidase II, encoded by the gene bglu2, from thermo- tolerant yeast Pichia etchellsii into Escherichia coli is described. Cloning of the 7.3 kb BamHI/SalI yeast insert containing bglu2 in pUC18, which allowed for reverse orientation of the insert, resulted in better enzyme expression. Transformation of this plasmid into E. coli JM109 resulted in accumulation of the enzyme in periplasmic space. At 50oC, the highest hydrolytic activity of 1686 IU/g protein was obtained on sophorose. Batch and fed-batch techniques were employed for enzyme production in a 14 L bioreactor. Exponential feeding rates were determined from mass balance equations and these were employed to control specific growth rate and in turn maximize cell growth and enzyme production. Media optimization coupled with this strategy resulted in increased enzyme units of 1.2 kU/L at a stabilized growth rate of 0.14 h-1. Increased enzyme production in bioreactor was accompanied by formation of inclusion bodies.

Cloning, Characterization of Pichia etchellsii $\beta-Glucosidase$ II and Effect of Media Composition and Feeding Strategy on its Production in a Bioreactor

Sethi Benu,Jain Monika,Chowdhary Manish,Soni Yogesh,Bhatia Yukti,Sahai Vikram,Mishra Saroj The Korean Society for Biotechnology and Bioengine 2002 Biotechnology and Bioprocess Engineering Vol.7 No.1

The cloning and expression of $\beta-glucosidase$ II, encoded by the gene ${\beta}glu2$, from thermotolerant yeast Pichia etchellsii into Escherichia coli is described. Cloning of the 7.3 kb BamHI/SalI yeast insert containing ${\beta}glu2$ in pUC18, which allowed for reverse orientation of the insert, resulted in better enzyme expression. Transformation of this plasmid into E. coli JM109 resulted in accumulation of the enzyme in periplasmic space. At $50^{\circ}C$, the highest hydrolytic activity of 1686 IU/g protein was obtained on sophorose. Batch and fed-batch techniques were employed for enzyme production in a 14 L bioreactor. Exponential feeding rates were determined from mass balance equations and these were employed to control specific growth rate and in turn maximize cell growth and enzyme production. Media optimization coupled with this strategy resulted in increased enzyme units of 1.2 kU/L at a stabilized growth rate of $0.14\;h^{-l}$. Increased enzyme production in bioreactor was accompanied by formation of inclusion bodies.

Comparison of propofol alone and in combination with ketamine or fentanyl for sedation in endoscopic ultrasonography

Shweta A Singh,Kelika Prakash,Sandeep Sharma,Gaurav Dhakate,Vikram Bhatia 대한마취통증의학회 2018 Korean Journal of Anesthesiology Vol.71 No.1

Background: We evaluated whether the addition of a small dose of ketamine or fentanyl would lead to a reduction in the total dose of propofol consumed without compromising the safety and recovery of patients having endoscopic ultrasonography (EUS). Methods: A total of 210 adult patients undergoing elective EUS under sedation were included in the study. Patients were randomized into three groups. Patients were premedicated intravenously with normal saline in group 1, 50 μg fentanyl in group 2, and 0.5 mg/kg ketamine in group 3. All patients received intravenous propofol for sedation. Propofol consumption in mg/kg/h was noted. The incidence of hypotension, bradycardia, desaturation, and coughing was noted. The time to achieve a Post Anesthesia Discharge Score (PADS) of 10 was also noted. Results: There were 68 patients in group 1, 70 in group 2, and 72 in group 3. The amount of propofol consumed was significantly higher in group 1 (9.25 [7.3–13.2]) than in group 2 (8.8 [6.8–12.2]) and group 3 (7.6 [5.7–9.8]). Patient hemodynamics and oxygenation were well maintained and comparable in all groups. The time to achieve a PADS of 10 was significantly higher in group 3 compared to the other two groups. Conclusions: The use of 50 μg fentanyl or 0.5 mg/kg ketamine in a single dose during EUS reduces the dose of propofol required for sedation. However, unlike the addition of fentanyl, the addition of ketamine increased the time to recovery. Thus, 50 μg fentanyl is a good additive to propofol infusion for sedation during EUS to reduce the requirement for propofol without affecting the time to recovery.

Endoscopic Ultrasound Can Differentiate High-Grade Pancreatic Intraepithelial Neoplasia, Small Pancreatic Ductal Adenocarcinoma, and Benign Stenosis

Sagami Ryota,Yamao Kentaro,Minami Ryuki,Nakahodo Jun,Akiyama Hidetoshi,Nishikiori Hidefumi,Mizukami Kazuhiro,Yamao Kenji,Bhatia Vikram,Amano Yuji,Murakami Kazunari 거트앤리버 소화기연관학회협의회 2024 Gut and Liver Vol.18 No.2

Background/Aims: High-grade pancreatic intraepithelial neoplasia and invasive pancreatic ductal adenocarcinoma ≤10 mm are targets for early detection of pancreatic cancer. However, their imaging characteristics are unknown. We aimed to identify endoscopic ultrasound findings for the detection of these lesions. Methods: Patients diagnosed with high-grade pancreatic intraepithelial neoplasia (n=29), pancreatic ductal adenocarcinoma ≤10 mm (n=11) (who underwent surgical resection), or benign main pancreatic duct stenosis (n=20) between January 2014 and January 2021 were retrospectively included. Six features differentiating these lesions were examined by endoscopic ultrasonography: main pancreatic duct stenosis, upstream main pancreatic duct dilation, hypoechoic areas surrounding the main pancreatic duct irregularities (mottled areas without demarcation or round areas with demarcation), branch duct dilation, prominent lobular segmentation, and atrophy. Interobserver agreement was assessed by two independent observers. Results: Hypoechoic areas surrounding the main pancreatic duct irregularities were observed more frequently in high-grade pancreatic intraepithelial neoplasia (82.8%) and pancreatic ductal adenocarcinoma ≤10 mm (90.9%) than in benign stenosis (15.0%) (p<0.001). High-grade pancreatic intraepithelial neoplasia exhibited mottled hypoechoic areas more frequently (79.3% vs 18.9%, p<0.001), and round hypoechoic areas less frequently (3.4% vs 72.7%, p<0.001), than pancreatic ductal adenocarcinoma ≤10 mm. The sensitivity and specificity of hypoechoic areas for differentiating high-grade pancreatic intraepithelial neoplasia, pancreatic ductal adenocarcinoma ≤10 mm, and benign stenosis were both 85.0%, with moderate interobserver agreement. Conclusions: The hypoechoic areas surrounding main pancreatic duct irregularities on endoscopic ultrasound may differentiate between high-grade pancreatic intraepithelial neoplasia, pancreatic ductal adenocarcinoma ≤10 mm, and benign stenosis (Trial Registration: UMIN Clinical Trials Registry (UMIN000044789).

Review : EUS-Guided Biliary Drainage

( Kenji Yamao ),( Kazuo Hara ),( Nobumasa Mizuno ),( Akira Sawaki ),( Susumu Hijioka ),( Yasumasa Niwa ),( Masahiro Tajika ),( Hiroki Kawai ),( Shinya Kondo ),( Yasuhiro Shimizu ),( Vikram Bhatia ) The Editorial Office of Gut and Liver 2010 Gut and Liver Vol.4 No.s1

Endoscopic ultrasonography (EUS) combines endoscopy and intraluminal ultrasonography, and allows imaging with a high-frequency transducer over a short distance to generate high-resolution ultrasonographic images. EUS is now a widely accepted modality for diagnosing pancreatobiliary diseases. EUS-guided fineneedle aspiration (EUS-FNA) using a curved linear- array echoendoscope was initially described more than 20 years ago, and since then many researchers have expanded its indications to sample diverse lesions and have also used it for various therapeutic purposes. EUS-guided biliary drainage (EUS-BD) is one of the therapeutic procedures that has been developed using a curved linear-array echoendoscope. Technically, EUS-BD includes rendezvous techniques via transesophageal, transgastric, and transduodenal routes, EUS-guided choledochoduodenostomy (EUS-CDS), and EUS-guided hepaticogastrostomy (EUS-HGS). Published data have demonstrated a high success rate, albeit with a comparatively high rate of nonfatal complications for EUS-CDS and EUS-HGS, and a comparatively low success rate with a low complication rate for the rendezvous technique. At present, these procedures represent an alternative to surgery or percutaneous transhepatic biliary drainage (PTBD) for patients with obstructive jaundice when endoscopic biliary drainage (EBD) has failed. However, these procedures should be performed in centers with extensive experience in linear EUS and therapeutic biliary ERCP. Large prospective studies are needed in the near future to establish standardized EUS-BD procedures as well as to perform controlled comparative trials between EUS-BD and PTBD, between rendezvous techniques and direct-access techniques (EUS-CDS and EUS-HGS), and between EBD and EUS-BD. (Gut Liver 2010;4(Suppl. 1):S67-75)