Development of an Anti-HER2 Single-Chain Variable Antibody Fragment Construct for High-Yield Soluble Expression in Escherichia coli and One-Step Chromatographic Purification.

Although single-chain variable fragment (scFv) is recognized as a highly versatile scaffold of recombinant antibody fragment molecules, its overexpression in Escherichia coli often leads to the formation of inclusion bodies. To address this issue, we devised and tested four different constructs, named v21, v22, v23 and v24, for producing anti-human epidermal growth factor receptor 2 (HER2) scFv. Among them, the v24 construct obtained from N-terminal fusion of maltose-binding protein (MBP) and subsequent tobacco etch virus protease (TEV) was identified as the most efficient construct for the production of anti-HER2 scFv. Aided by an MBP tag, high-yield soluble expression was ensured and soluble scFv was liberated in cells via autonomous proteolytic cleavage by endogenously expressed TEV. The isolated scFv containing a C-terminal hexahistidine tag was purified through a one-step purification via nickel-affinity chromatography. The purified scFv exhibited a strong (nanomolar Kd) affinity to HER2 both in vitro and in cells. Structural and functional stabilities of the scFv during storage for more than one month were also assured. Given the great utility of anti-HER2 scFv as a basic platform for developing therapeutic and diagnostic agents for cancers, the v24 construct and methods presented in this study are expected to provide a better manufacturing system for producing anti-HER2 scFv with various industrial applications.

Design and Prediction of Aptamers Assisted by In Silico Methods.

An aptamer is a single-stranded DNA or RNA that binds to a specific target with high binding affinity. Aptamers are developed through the process of systematic evolution of ligands by exponential enrichment (SELEX), which is repeated to increase the binding power and specificity. However, the SELEX process is time-consuming, and the characterization of aptamer candidates selected through it requires additional effort. Here, we describe in silico methods in order to suggest the most efficient way to develop aptamers and minimize the laborious effort required to screen and optimise aptamers. We investigated several methods for the estimation of aptamer-target molecule binding through conformational structure prediction, molecular docking, and molecular dynamic simulation. In addition, examples of machine learning and deep learning technologies used to predict the binding of targets and ligands in the development of new drugs are introduced. This review will be helpful in the development and application of in silico aptamer screening and characterization.

Papillary Thyroid Cancer Tumor Spheres Cultured by Passaging Without Sorting Exhibit Cancer Stemness.

AIM: Cancer stem-like cell (CSC) markers and the role of CSCs derived from papillary thyroid carcinoma (PTC) in pathogenesis are unclear. This study aimed to investigate CSC properties using tumor spheres from passaged PTC cells but without sorting CSCs. MATERIALS AND METHODS: To identify the properties of CSCs derived from PTC, the expression of SRY-box transcription factor 2(SOX2), octamer-binding transcription factor 4 (OCT4), Nanog homeobox (NANOG), thyroglobulin (TG), thyroid-stimulating hormone receptor (TSHR), E-cadherin, YES-associated protein 1 (YAP1), and signal transducer and activator of transcription 3 (STAT3) was investigated in tumor spheres serially passaged without sorting CSCs. RESULTS: The cultured tumor spheres had cancer stemness; high expression of OCT4, SOX2, NANOG, and YAP1; low expression of E-cadherin; and varied expression of TG, TSHR, and STAT3. PTC tumor spheres transfected with small interfering RNA targeting YAP1 had fewer CSC properties than the non-transfected tumor spheres did. CONCLUSION: Tumor spheres derived from PTC cells by passaging without sorting CSCs have more stem-like cell properties, and less differentiation potential. Thus, this simple and cost-effective method can be used for the enrichment of PTC stemness for employment in cell-based models, reducing the need for use of animal models.

Effect of sleeve gastrectomy on type 2 diabetes as an alternative treatment modality to Roux-en-Y gastric bypass: systemic review and meta-analysis.

BACKGROUND: Until recently, Roux-en-Y gastric bypass (RYGB) was the most frequently performed procedure in bariatric surgery. In the last decade, sleeve gastrectomy (SG) has emerged as a more popular, simpler, and less morbid form of bariatric surgery. OBJECTIVES: This study compares the efficacy of SG and RYGB for the treatment of type 2 diabetes mellitus (T2D). SETTING: Systemic review and meta-analysis. METHODS: MEDLINE, EMBASE, and the Cochrane Library were searched for entries up to December 2013. Search terms included "Sleeve gastrectomy," "Gastric bypass," and "Type 2 diabetes mellitus." The chosen articles described both "Sleeve gastrectomy" and "Gastric bypass" and included over 1 year of follow-up data. Data analysis was performed with Review Manager 5.2 and SPSS version 20. RESULTS: The data set is comprised of 3 retrospective clinical studies, 6 prospective clinical studies, and 2 randomized controlled trials (RCTs), which involved 429 patients in the SG group and 428 patients in the RYGB group. In nonrandomized clinical studies, SG displayed similar efficacy in remission of T2D compared with the standard RYGB. In the RCTs, SG had a lower effect than that of RYGB. T2D remission was not correlated with the percent of excess weight loss for either procedure. CONCLUSIONS: Based on the current evidence, SG has a similar effect on T2D remission as RYGB.

Should lymph node micrometastasis be considered in node staging for gastric cancer?: the significance of lymph node micrometastasis in gastric cancer.

BACKGROUND: In TNM staging system, lymph node staging is based on the number of metastatic lymph nodes in gastric cancer and micrometastasis is not considered. Several reports proposed the importance of lymph node micrometastasis as the causative factor for recurrence and poor survival, but it remains controversial among researchers. METHODS: A total of 482 gastric cancer patients who underwent curative resection from 2004 to 2010 at Korea University Medical Center Ansan Hospital, South Korea were prospectively enrolled. For detecting lymph node micrometastasis, immunohistochemical staining with anti-cytokeratin antibody (CAM 5.2) was performed on negative lymph nodes by hematoxylin-eosin (H-E) staining. Survival differences were compared between conventional node staging and new node staging that took micrometastasis into consideration. Also, the prognostic value of lymph node micrometastasis was investigated in multivariate analysis. RESULTS: A total of 156 patients (32.4%) showed lymph node micrometastasis. Overall, the micrometastatic group had more advanced tumor and lymph node stage, lymphovascular cancer cell invasion, a higher rate of recurrence, and poor survival. Furthermore, when the cumulative numbers of macro- and micrometastatic lymph nodes were calculated together, the discriminative power of survival difference between each node stage became more stratified. Also, multivariate analysis using Cox's proportional hazards model demonstrated perineural invasion, pathologic T stage, dissected lymph nodes, macro- and micrometastatic lymph nodes are independent prognostic factors. CONCLUSIONS: Lymph node micrometastasis was clinically significant as a risk factor for recurrent gastric cancer. Lymph node micrometastasis should be considered when estimating TNM stage for determining prognosis and the best treatment strategy.

Pattern, Timing and Survival in Patients with Recurrent Gastric Cancer.

BACKGROUND/AIMS: The aim of this study was to investigate the recurrence patterns, the timing of recurrence, and the survival rate in recurrent cases of gastric cancer. METHODOLOGY: Of 1,029 patients who underwent curative resection for gastric cancer at the Department of Surgery, Korea University Guro Hospital between 2000 and 2006, 146 patients developed recurrence and were included in this study. Timing and patterns of recurrence, the recurrence pattern according to clinicopathological factors, and post-recurrence survival rate were analyzed retrospectively. RESULTS: The mean time to recurrence was 21.2 months. Forty-two patients (28.8%) had recurrence within 1 year, and 54 patients (37.0%) had recurrence 1-2 years after surgery. Single-site recurrence occurred in 72.6% of patients, and multiple-site recurrence in 27.4%. The most frequent pattern of recurrence was peritoneal recurrence in 39.7% of patients, hematogeneous in 24.7%, locoregional in 18.5%, and to a distant lymph node in 17.1%. In cases that showed recurrence within 1 year, the most frequent pattern of recurrence was hematogeneous recurrence, while it was peritoneal in the group with recurrence between 1 and 2 years after surgery. Patterns of recurrence significantly differed according to the sex and gross tumor morphology. The mean post-recurrence survival time was 15.7 months. There was no statistically significant difference in the post-recurrence survival time according to the pattern of recurrence. CONCLUSIONS: The most frequent pattern of recurrence was peritoneal recurrence, and recurrence most often occurred within 2 years after curative resection. There was no significant difference in post-recurrence survival time according to the pattern of recurrence.

Minimizing operative time for robotic gastrectomy in cancer: analysis of the major factors for four detailed steps.

BACKGROUND/AIMS: We outlined the major factors for minimizing the operative time of robotic-assisted gastrectomy (RAG) during the initial learning period. METHODOLOGY: We performed correlation analysis and multivariate linear regression for detailed operation steps including preparing, docking, console and anastomosis time. RESULTS: Forty patients underwent RAG for cancer. By Pearson analysis, case number (r = -0.313; P = 0.049) and body mass index (BMI) (r = 0.368; P = 0.019) were found to be correlated with total operation time. Multivariate linear regression with backward elimination showed that BMI and case number significantly affected total operation time. A detailed four-step analysis showed that docking time was significantly affected by intraoperative complications. CONCLUSIONS: In conclusion, we recommend a V-shape port placement as an important surgical factor for preparation and docking time to avoid unnecessary intraoperative trial errors. In addition, selecting lower-BMI patients would be helpful in shortening the time to mastery of console, which is the most time consuming operative step of the robotic procedure for robotic-naive surgeons.

Primary gastric malignant melanoma mimicking adenocarcinoma.

We report a case of primary gastric malignant melanoma that was diagnosed after curative resection but initially misdiagnosed as adenocarcinoma. A 68-year-old woman was referred to our department for surgery for gastric adenocarcinoma presenting as a polypoid lesion with central ulceration located in the upper body of the stomach. The preoperative diagnosis was confirmed by endoscopic biopsy. We performed laparoscopic total gastrectomy, and the final pathologic evaluation led to the diagnosis of primary gastric malignant melanoma without a primary lesion detected in the body. To the best of our knowledge, primary gastric malignant melanoma is extremely rare, and this is the first case reported in our country. According to the literature, it has aggressive biologic activity compared with adenocarcinoma, and curative resection is the only promising treatment strategy. In our case, the patient received an early diagnosis and underwent curative gastrectomy with radical lymphadenectomy, and no recurrence was noted for about two years.