Exploring the potential of Bacillus subtilis as cell factory for food ingredients and special chemicals.

BACKGROUND: Bacillus subtilis has been established as model microorganism for fundamental research in the laboratory on protein production/secretion and sporulation and as model bacterium for controlling spoilage in the food industry. It has also been used for production of (commercial) enzymes and several secondary metabolites such as vitamins. However, this doesn't fully reflect the potential of B. subtilis as a cell-factory. Here, various strains of B. subtilis, including food-grade, spore-deficient strains and industrially used strains, were compared for their growth and metabolic potential. Industry-relevant parameters were analyzed for all strains under various aeration regimes, under anaerobic conditions, in various nutritious and nutrient-limited cultivation media, with and without organic nitrogen sources, and with and without sugar. RESULTS: Practical experiments were conducted to compare industrial relevant properties like growth rates, intracellular components and extracellular metabolite profile of different B. subtilis strains. Based on growth flexibility in different media, we found that some strains like NCIB3610 and DSM1092 are adapted to inorganic or organic nitrogen source utilization, which is highly relevant when considering a biorefinery approach using various cheap and abundant waste/sidestreams. Secondly, spore-deficient strains such as 3NA, 168 S and PY79S, showed advantages in microbial protein and acetolactate pathway expression, which is associated with applications in food industry for protein supplement and diacetyl production. Lastly, WB800 and PY79S exhibited potential for fermentative production of dipicolinic acid, 2,3-butanediol and lactic acid that could serve as precursors for biopolymers. CONCLUSION: This study demonstrates the broad potential for more extensive industrial use of Bacillus subtilis in the (bio-based) chemical industry for use of sidestreams, in the personal care industry, in the food industry for food additive production, and in the bio-sustainable industry for biofuel and bio-degradable plastic precursors production. In addition, selecting different B. subtilis strains for specific purposes makes full use of the diversity of this species and increases the potential of B. subtilis in its contribution to the bio-based economy.

Multilayer Genetic Circuits for Dynamic Regulation of Metabolic Pathways.

The dynamic regulation of metabolic pathways is based on changes in external signals and endogenous changes in gene expression levels and has extensive applications in the field of synthetic biology and metabolic engineering. However, achieving dynamic control is not trivial, and dynamic control is difficult to obtain using simple, single-level, control strategies because they are often affected by native regulatory networks. Therefore, synthetic biologists usually apply the concept of logic gates to build more complex and multilayer genetic circuits that can process various signals and direct the metabolic flux toward the synthesis of the molecules of interest. In this review, we first summarize the applications of dynamic regulatory systems and genetic circuits and then discuss how to design multilayer genetic circuits to achieve the optimal control of metabolic fluxes in living cells.

Design and construction of novel biocatalyst for bioprocessing: Recent advances and future outlook.

Bioprocess, a biocatalysis-based technology, is becoming popular in many research fields and widely applied in industrial manufacturing. However, low bioconversion, low productivity, and high costs during industrial processes are usually the limitation in bioprocess. Therefore, many biocatalyst strategies have been developed to meet these challenges in recent years. In this review, we firstly discuss protein engineering strategies, which are emerged for improving the biocatalysis activity of biocatalysts. Then, we summarize metabolic engineering strategies that are promoting the development of microbial cell factories. Next, we illustrate the necessity of using the combining strategy of protein engineering and metabolic engineering for efficient biocatalysts. Lastly, future perspectives about the development and application of novel biocatalyst strategies are discussed. This review provides theoretical guidance for the development of efficient, sustainable, and economical bioprocesses mediated by novel biocatalysts.

Combinatorial Methylerythritol Phosphate Pathway Engineering and Process Optimization for Increased Menaquinone-7 Synthesis in Bacillus subtilis.

Vitamin K2 (menaquinone) is an essential vitamin existing in the daily diet, and menaquinone-7 (MK-7) is an important form of it. In a recent work, we engineered the synthesis modules of MK-7 in Bacillus subtilis, and the strain BS20 could produce 360 mg/l MK-7 in shake flasks, while the methylerythritol phosphate (MEP) pathway, which provides the precursor isopentenyl diphosphate for MK-7 synthesis, was not engineered. In this study, we overexpressed five genes of the MEP pathway in BS20 and finally obtained a strain (BS20DFHG) with MK-7 titer of 415 mg/l in shake flasks. Next, we optimized the fermentation process parameters (initial pH, temperature and aeration) in an 8-unit parallel bioreactor system consisting of 300-ml glass vessels. Based on this, we scaled up the MK-7 production by the strain BS20DFHG in a 50-l bioreactor, and the highest MK-7 titer reached 242 mg/l. Here, we show that the engineered strain BS20DFHG may be used for the industrial production of MK-7 in the future.

Cell Membrane and Electron Transfer Engineering for Improved Synthesis of Menaquinone-7 in Bacillus subtilis.

The formation of biofilm facilitates the synthesis of valuable natural product menaquinone-7 (MK-7) in static culture of Bacillus subtilis, whereas the essential role and mechanism of biofilm in MK-7 synthesis have not been revealed. Herein, comparative transcriptomics show that the formation of biofilm affected MK-7 synthesis by changing the transcription levels of signal receptor (BSU02010), transmembrane transporter (BSU29340, BSU03070), and signal transduction (BSU02630). Moreover, we also found that oxalate decarboxylase OxdC has an important effect on electron generation and MK-7 synthesis, when the transcriptional level of NADH dehydrogenase decreases in static culture. Our results revealed that cell membrane and electron transfer are important factors in promoting MK-7 synthesis.

Design of a programmable biosensor-CRISPRi genetic circuits for dynamic and autonomous dual-control of metabolic flux in Bacillus subtilis.

Dynamic regulation is an effective strategy for fine-tuning metabolic pathways in order to maximize target product synthesis. However, achieving dynamic and autonomous up- and down-regulation of the metabolic modules of interest simultaneously, still remains a great challenge. In this work, we created an autonomous dual-control (ADC) system, by combining CRISPRi-based NOT gates with novel biosensors of a key metabolite in the pathway of interest. By sensing the levels of the intermediate glucosamine-6-phosphate (GlcN6P) and self-adjusting the expression levels of the target genes accordingly with the GlcN6P biosensor and ADC system enabled feedback circuits, the metabolic flux towards the production of the high value nutraceutical N-acetylglucosamine (GlcNAc) could be balanced and optimized in Bacillus subtilis. As a result, the GlcNAc titer in a 15-l fed-batch bioreactor increased from 59.9 g/l to 97.1 g/l with acetoin production and 81.7 g/l to 131.6 g/l without acetoin production, indicating the robustness and stability of the synthetic circuits in a large bioreactor system. Remarkably, this self-regulatory methodology does not require any external level of control such as the use of inducer molecules or switching fermentation/environmental conditions. Moreover, the proposed programmable genetic circuits may be expanded to engineer other microbial cells and metabolic pathways.

Clinicopathologic and Prognostic Significance of Thymopoietin-α Overexpression in Gastric Cancer.

As one of the deadliest and most common malignancies in the world, gastric cancer (GC) represents a serious health threat. Despite recent advances in the field, the prognosis of patients with metastatic GC remains poor. In this study, we aimed to investigate the clinical impact of the alpha subunit of the nuclear structural protein thymopoietin (TMPO-α) in GC. The expression of TMPO-α in seven gastric cell lines was detected by immunoblotting. The expression level of TMPO-α levels in gastric tissues collected from 145 GC patients was examined by immunohistochemistry. The correlations between TMPO-α expression level and clinicopathologic parameters, as well as the association of TMPO-α expression with overall survival, were assessed. Immunohistochemistry showed that the expression of TMPO-α was significantly higher in GC tissues and cells in comparison with non-tumor tissues and cells. Furthermore, the overexpression of TMPO-α in gastric tissues (56%) was positively associated with Lauren classification (P = 0.0159), nodal status (P = 0.0265), distant metastasis (P < 0.0001), stage (P = 0.0367), and degree of differentiation (P = 0.0009). Patients with high TMPO-α levels had a significantly poorer overall survival than those with low levels (P = 0.001). Multivariate Cox regression analysis also indicated that TMPO-α was an independent prognostic marker for GC (P = 0.045). In addition, studies conducted in GC cells indicated that knockdown of TMPO-α suppressed cell proliferation and invasion. These findings indicate that TMPO-α overexpression can predict clinicopathologic features and the outcome of patients with GC.

Engineering the Substrate Transport and Cofactor Regeneration Systems for Enhancing 2'-Fucosyllactose Synthesis in Bacillus subtilis.

Human milk oligosaccharides (HMOs) have been proven to be beneficial to infants' intestinal health and immune systems. 2'-Fucosyllactose (2'-FL) is the most abundant and thoroughly studied HMO and has been approved to be an additive of infant formula. How to construct efficient and safe microbial cell factories for the production of 2'-FL attracts increasing attention. In this work, we engineered the Bacillus subtilis as an efficient 2'-FL producer by engineering the substrate transport and cofactor guanosine 5'-triphosphate (GTP) regeneration systems. First, we constructed a synthesis pathway for the 2'-FL precursor guanosine 5'-diphosphate-l-fucose (GDP-l-fucose) by introducing the salvage pathway gene fkp from Bacteriodes fragilis and improved the fucose importation by overexpressing the transporters. Then, the complete synthesis pathway of 2'-FL was constructed by introducing the heterologous fucosyltransferases from different sources, and it was found that the gene from Helicobacter pylori was the best one for 2'-FL synthesis. We also improved the substrate lactose importation by introducing heterologous lactose permeases and eliminated endogenous ß-galactosidase (yesZ) to block the lactose degradation. Next, the production of 2'-FL and GDP-l-fucose was improved by fine-tuning the expression of cofactor guanosine 5'-triphosphate regeneration module genes gmd, ndk, guaA, guaC, ykfN, deoD, and xpt. Finally, a 3 L fed-batch fermentation was performed, and the highest 2'-FL titer reached 5.01 g/L with a yield up to 0.85 mol/mol fucose. We optimized the synthesis modules of 2'-FL in B. subtilis, and this provides a good starting point for metabolic engineering to further improve 2'-FL production in the future.

CRISPRi allows optimal temporal control of N-acetylglucosamine bioproduction by a dynamic coordination of glucose and xylose metabolism in Bacillus subtilis.

Glucose and xylose are the two most abundant sugars in renewable lignocellulose sources; however, typically they cannot be simultaneously utilized due to carbon catabolite repression. N-acetylglucosamine (GlcNAc) is a typical nutraceutical and has many applications in the field of healthcare. Here, we have developed a gene repressor system based on xylose-induced CRISPR interference (CRISPRi) in Bacillus subtilis, aimed at downregulating the expression of three genes (zwf, pfkA, glmM) that control the major competing reactions of GlcNAc synthesis (pentose phosphate pathway (HMP), glycolysis, and peptidoglycan synthesis pathway (PSP)), with the potential to relieve glucose repression and allow the co-utilization of both glucose and xylose. Simultaneous repression of these three genes by CRISPRi improved GlcNAc titer by 13.2% to 17.4 ±â€¯0.47 g/L, with the GlcNAc yield on glucose and xylose showing an 84.1% improvement, reaching 0.42 ±â€¯0.036 g/g. In order to further engineer the synergetic utilization of glucose and xylose, a combinatorial approach was developed based on 27 arrays containing sgRNAs with different repression capacities targeting the three genes. We further optimized the temporal control of the system and found that when 15 g/L xylose was added 6 h after inoculation, the most efficient strain, BNX122, synthesized 20.5 ±â€¯0.85 g/L GlcNAc with a yield of 0.46 ±â€¯0.010 g/g glucose and xylose in shake flask culture. Finally, the GlcNAc titer and productivity in a 3-L fed-batch bioreactor reached 103.1 ±â€¯2.11 g/L and 1.17 ±â€¯0.024 g/L/h, which were 5.0-fold and 2.7-fold of that in shake flask culture, respectively. Taken together, these findings suggest that a CRISPRi-enabled regulation method provides a simple, efficient, and universal way to promote the synergetic utilization of multiple carbon sources by microbial cell factories.

Impacts of autophagy-inducing ingredient of areca nut on tumor cells.

Areca nut (AN) is a popular carcinogen used by about 0.6-1.2 billion people worldwide. Although AN contains apoptosis-inducing ingredients, we previously demonstrated that both AN extract (ANE) and its 30-100 kDa fraction (ANE 30-100K) predominantly induce autophagic cell death in both normal and malignant cells. In this study, we further explored the action mechanism of ANE 30-100K-induced autophagy (AIA) in Jurkat T lymphocytes and carcinoma cell lines including OECM-1 (mouth), CE81T/VGH (esophagus), SCC25 (tongue), and SCC-15 (tongue). The results showed that chemical- and small hairpin RNA (shRNA)-mediated inhibition of AMP-activated protein kinase (AMPK) resulted in the attenuation of AIA in Jurkat T but not in OECM-1 cells. Knockdown of Atg5 and Beclin 1 expressions ameliorated AIA in OECM-1/CE81T/VGH/Jurkat T and OECM-1/SCC25/SCC-15, respectively. Furthermore, ANE 30-100K could activate caspase-3 after inhibition of Beclin 1 expression in OECM-1/SCC25/SCC15 cells. Meanwhile, AMPK was demonstrated to be the upstream activator of the extracellular-regulated kinase (ERK) in Jurkat T cells, and inhibition of MEK attenuated AIA in Jurkat T/OECM-1/CE81T/VGH cells. Finally, we also found that multiple myeloma RPMI8226, lymphoma U937, and SCC15 cells survived from long-term non-cytotoxic ANE 30-100K treatment exhibited stronger resistance against serum deprivation through upregulated autophagy. Collectively, our studies indicate that Beclin-1 and Atg5 but not AMPK are commonly required for AIA, and MEK/ERK pathway is involved in AIA. Meanwhile, it is also suggested that long-term AN usage might increase the resistance of survived tumor cells against serum-limited conditions.

α2-Adrenoceptor agonist dexmedetomidine protects septic acute kidney injury through increasing BMP-7 and inhibiting HDAC2 and HDAC5.

Bone morphogenetic protein (BMP)-7 protects sepsis-induced acute kidney injury (AKI). Dexmedetomidine (DEX), an α(2)-adrenoceptor (α(2)-AR) agonist, has anti-inflammatory effects. We investigated the protective effects of DEX on sepsis-induced AKI and the expression of BMP-7 and histone deacetylases (HDACs). In vitro, the effects of DEX or trichostatin A (TSA, an HDAC inhibitor) on TNF-α, monocyte chemotactic protein (MCP-1), BMP-7, and HDAC mRNA expression in LPS-stimulated rat renal tubular epithelial NRK52E cells, was determined using real-time PCR. In vivo, mice were intraperitoneally injected with DEX (25 µg/kg) or saline immediately and 12 h after cecal ligation and puncture (CLP) surgery. Twenty-four hours after CLP, we examined kidney injury and renal TNF-α, MCP-1, BMP-7, and HDAC expression. Survival was monitored for 120 h. LPS increased HDAC2, HDAC5, TNF-α, and MCP-1 expression, but decreased BMP-7 expression in NRK52E cells. DEX treatment decreased the HDAC2, HDAC5, TNF-α, and MCP-1 expression, but increased BMP-7 and acetyl histone H3 expression, whose effects were blocked by yohimbine, an α(2)-AR antagonist. With DEX treatment, the LPS-induced TNF-α expression and cell death were attenuated in scRNAi-NRK52E but not BMP-7 RNAi-NRK52E cells. In CLP mice, DEX treatment increased survival and attenuated AKI. The expression of HDAC2, HDAC5, TNF-α, and MCP-1 mRNA in the kidneys of CLP mice was increased, but BMP-7 was decreased. However, DEX treatment reduced those changes. DEX reduces sepsis-induced AKI by decreasing TNF-α and MCP-1 and increasing BMP-7, which is associated with decreasing HDAC2 and HDAC5, as well as increasing acetyl histone H3.

Possible mechanism of betel-quid-extract-induced expression of matrix metalloproteinase-2.

BACKGROUND/PURPOSE: Betel quid extract (BQE) has been demonstrated to induce matrix metalloproteinase (MMP)-2 expression. This study aimed to establish the possible mechanism involved in this event. METHODS: Western blotting, reverse-transcription polymerase chain reaction, and gelatin zymography were used to study the expression level of MMP-2. LY294002, PD98059, U0126, N-acetyl-L-cysteine, SB203580, SP600125, and Bay 11-7082 were used to pretreat OECM-1 cells before BQE treatment and MMP-2 detection. RESULTS: OECM-1 cells were subjected to short-term (10 minutes) or long-term (24 hours) BQE treatment (designated as SBT and LBT, respectively), and we found that both treatments increased MMP-2 protein and extracellular signal-regulated kinase (ERK) phosphorylation levels in a concentration- and time-dependent manner. LBT also increased MMP-2 mRNA level. LBT-induced MMP-2 secretion was not inhibited by PD98059 (up to 50 µM) when ERK was effectively blocked, but was attenuated by LY294002 (0-10 µM) in a concentration-dependent manner. This LBT effect was inhibited strongly by SB203580 (10 µM), SP600125 (10 µM), and Bay 11-7082 (10 µM) and mildly by N-acetyl-L-cysteine (5 mM), but not by U0126 (10 µM). CONCLUSION: Both SBT and LBT upregulate MMP-2 expression, and LBT-induced MMP-2 expression might be mediated by phosphoinositide 3-kinase, p38 mitogen-activated protein kinase, c-Jun N-terminal kinase, and nuclear factor-κB, and to a lesser extent, by reactive oxygen species, rather than by ERK.

Autophagy induction by the 30-100kDa fraction of areca nut in both normal and malignant cells through reactive oxygen species.

Areca nut (AN) is an addictive carcinogen used by about 200-600 million people worldwide. Some AN components are shown to induce apoptosis; however, we previously demonstrated that AN extract (ANE) and the 30-100kDa fraction of ANE (ANE 30-100K) induced autophagy-like responses, such as swollen cell morphology, empty cytoplasm, acidic vesicles, and LC3-II accumulation, in an oral cancer cell line, OECM-1. To further assess the responses of other cell types to ANE 30-100K, we used both normal and malignant cells as the targets of ANE 30-100K and found that normal oral fibroblasts (CMT415), peripheral blood lymphocytes (PBLs), Jurkat leukemia T cells, and esophageal carcinoma cells (CE81T/VGH) exhibited similar responses after ANE 30-100K challenge. ANE 30-100K drastically increased acidic vesicle-containing PBLs isolated from two independent donors (from 0.1% to 92.1% and 2.9% to 64.2%). Furthermore, both ANE- and ANE 30-100K-induced LC3-II accumulation in CMT415 and CE81T/VGH was further increased in the presence of the lysosomal protease inhibitors (pepstatin A, E64d, and leupeptin). On the other hand, ANE 30-100K also increased the level of intracellular reactive oxygen species (ROS), and the ROS scavengers, N-acetylcysteine (NAC) and Tiron, inhibited ANE 30-100K-induced cell death and LC3-II accumulation. Collectively, these results suggest the existence of an autophagy-inducing AN ingredient (AIAI) in ANE 30-100K, which renders ANE as an autophagic flux inducer through ROS in both normal and malignant cells.

Totally laparoscopic radical BII gastrectomy for the treatment of gastric cancer: a comparison with open surgery.

BACKGROUND: Laparoscopically assisted distal gastrectomy has been used for distal part early gastric cancer resection. However, use of totally laparoscopic gastric cancer resection remains limited because of technical problems, especially when standard D2 nodal dissection was applied. We had reported the first totally laparoscopic Billroth II (BII) subtotal gastrectomy with lymphadenectomy for early gastric cancer in the year 1998. The aim of this study is to determine whether this procedure is superior to conventional open technique. METHODS: The clinical course of 34 consecutive patients who underwent totally laparoscopic BII gastrectomy using an upper to lower, right to left, and clockwise quadrant-to-quadrant technique was compared with 34 sex-matched and age-matched patients who underwent open gastrectomy. Main outcome measures included operative time, blood loss, length of stay, morbidity and mortality, adequacy of lymphadenectomy, and long-term outcome. RESULTS: In the laparoscopic group, all the operations were completed by laparoscopic technique, but 1 patient required secondary laparotomy for total gastrectomy owing to inadequate resection margin. There was no operation mortality in this study. The postoperative complication rates were similar in these 2 groups. The mean operative time for laparoscopic group was 283+/-122 minutes (range: 186 to 480 min), significantly longer than the 195+/-26 minutes in the conventional group (P<0.001). Laparoscopic group was associated with less intraoperative blood loss (74 vs. 190 mL; P<0.01), early flatus passage (2.9 vs. 4.9 d; P<0.01), less usage of analgesics (3.5 vs. 5.8 doses; P<0.05), and a shorter postoperative hospital stay (8.5 vs. 12.1 d; P<0.01). There was no significant difference between laparoscopic and conventional open radical gastrectomy with regard to ratio of free margin, number of harvested lymph nodes, and survival. CONCLUSION: Although totally laparoscopic BII gastrectomy using the upper to lower technique required a longer surgical time and was technically more demanding than conventional open surgery, it resulted in shorter recovery time, less analgesic use, and less severe physical discomfort without compromising the operative curability and oncologic outcomes.

Upregulation of matrix metalloproteinase-1 (MMP-1) expression in oral carcinomas of betel quid (BQ) users: roles of BQ ingredients in the acceleration of tumour cell motility through MMP-1.

Matrix metalloproteinases (MMPs) are commonly expressed in carcinomas including oral squamous cell carcinomas (OSCCs). On the other hand, some evidences suggested that ingredients of betel quid (BQ) inhibit the activity and/or expression of some MMPs thought to be the pathogenesis of oral submucous fibrosis. This study was to analyse whether MMP-1 expression is inhibited in OSCC specimens from BQ users and in cell lines survived from the challenge of BQ ingredients. We found that MMP-1 mRNA was expressed in all the tested 27 OSCC. Levels of MMP-1 mRNA and protein were significantly elevated in the tested five OSCC specimens than in their adjacent tissues (P<0.001 and 0.05, respectively). Esophageal carcinoma (CE81T/VGH) and OSCC (OECM-1) cell lines survived from the cytotoxic BQ extract (BQE) and arecoline selection process were found to express higher MMP-1 mRNA and protein levels, or to exhibit a significant acceleration of two-dimensional (2D) motility than their non-selected parental cells. The enhanced motility was further demonstrated to be specifically and significantly inhibited by the MMP-1 neutralizing antibody and/or by the transfection of an MMP-1 specific antisense oligodeoxynucleotide. These results suggest that in some carcinomas of the upper aerodigestive tract, BQ usage may upregulate MMP-1 expression in the survived tumour cells, and increase their mobility in an MMP-1-dependent manner.

Up-regulation of matrix metalloproteinase-8 by betel quid extract and arecoline and its role in 2D motility.

Betel quid (BQ) and matrix metalloproteinase-8 (MMP-8) play roles in oral diseases. Here, we analyzed the regulation of MMP-8 by BQ and its effect on cell migration. We found that BQ extract (BQE) increased the secretion of an 85kDa caseinolytic proteinase, specifically precipitated by an anti-MMP-8 antibody, in the culture medium of OECM-1, an oral squamous cell carcinoma (OSCC) cell line. BQE also stimulated MMP-8 secretion in an esophageal carcinoma cell line, CE81T/VGH, in a dose-dependent manner, and MMP-8 protein was maximally expressed at 24h after BQE treatment in OECM-1. The BQE-induced MMP-8 expression was dose-dependently inhibited by PD98059. Arecoline, the major alkaloid of areca nut, was tested to dose-dependently up-regulate MMP-8 protein level. Moreover, both arecoline- (4.7-fold) and BQE-selected (5.5-fold) CE81T/VGH cells expressed higher MMP-8 protein level and exhibited enhanced two-dimensional (2D) motility (p=0.009 in both cells) than parental cells. The enhanced motility of arecoline- (p=0.006) and BQE-selected (p=0.002) cells was both specifically blocked by an anti-MMP-8 antibody. We conclude that BQ may accelerate tumor migration by stimulating MMP-8 expression through MEK pathway in at least some carcinomas of the upper aerodigestive tract. Furthermore, arecoline may be one of the positive MMP-8 regulators among BQ ingredients.

Role of appendectomy in laparoscopic training.

PURPOSE: This study aimed to evaluate whether laparoscopic appendectomy is suitable for training residents to become proficient in laparoscopic surgery. MATERIALS AND METHODS: A total of 1574 laparoscopic appendectomies were performed at En-Chu-Kong Hospital between January 1998 and December 2003 (788 men and 786 women). These cases were divided into three groups: 543 cases (in 1998-1999) performed by 5 attending surgeons during the learning and trial stage; 536 cases (in 2000-2001) performed by 5 attending surgeons assisted by 2 senior residents with prior experience in open appendectomy; and 495 cases (in 2002-2003) done by these 2 senior residents, supervised by the attending surgeons. Demographic data, intraoperative findings, operative time, conversion rate, frequency of analgesic injection, timing of oral intake, hospital stay, morbidity, and mortality were analyzed. RESULTS: There were no statistically significant differences in the operations performed by attending surgeons (mature stage) vs. senior residents in terms of intraoperative findings, operative time (60.1 +/- 60.4 minutes vs. 56.3 +/- 25.6 minutes), conversion rate (1.12% vs. 0.81%), frequency of analgesic injection (0.57 +/- 1.37 times/stay vs. 0.43 +/- 0.94 times/stay), timing of oral intake (23.7 +/- 30.2 hours vs. 20.8 +/- 27.5 hours), hospital stay (73.9 +/- 61.8 hours vs. 70.3 +/- 51.6 hours), morbidity, or mortality (0% vs. 0.2%). CONCLUSION: Laparoscopic appendectomy can be safely incorporated into the training of surgical residents under the supervision of experienced surgeons. Laparoscopic appendectomy also provides knowledge of the basics of laparoscopic technique before going on to more complex operations.

Palliative gastrojejunostomy for advanced gastric antral cancer: double scope technique.

Gastrojejunostomy through laparotomy is a traditional procedure for the relief of gastric outlet obstruction in patients with unresectable carcinoma of the gastric antrum. However, patients face pain and high rates of morbidity and mortality after this operation. We present two cases of gastrojejunostomy for unresectable and obstructing gastric antral cancer using a double scope technique. The operative time was approximately 90 minutes for each patient, and the postoperative hospital stays were 9 and 10 days, respectively. Operative results were good. Vomiting was relieved. There were no delays in gastric emptying. No recurrence of gastric outlet obstruction was noted in the first patient, who died 9 months postoperatively. The second patient survived without this problem 6 months postoperatively. Palliative gastrojejunostomy via the double scope technique is a safe and effective minimally invasive procedure for the relief of gastric outlet obstruction in patients with unresectable carcinoma of the gastric antrum. It also provides accurate staging of the disease perioperatively. It should be considered the surgical choice for geriatric patients when clinically appropriate.

Colonoscopic-assisted laparoscopic resection of a colon lipoma.

We report the case of a 51-year-old man who had abdominal distension and intermittent cramping pain for 4 months. A lower gastrointestinal double-contrast study revealed a protruding mass at the transverse colon. Endoscopic ultrasonography showed a lesion arising from the submucosal layer. Instead of a laparotomy, the lesion was removed laparoscopically with the assistance of a colonoscope. No operative morbidity was noted. With future improvements of this technique, tumors that are difficult to remove with colonoscopy can be excised laparoscopically without the aid of an endoscopic stapler. In our case, the tumor was localized intraoperatively and extracted using the intraoperative colonoscopy. This double-scope technique is safe and cost-effective for some difficult colonic lesions.