Enhanced efficacy of glycoengineered rice cell-produced trastuzumab.

For several decades, a plant-based expression system has been proposed as an alternative platform for the production of biopharmaceuticals including therapeutic monoclonal antibodies (mAbs), but the immunogenicity concerns associated with plant-specific N-glycans attached in plant-based biopharmaceuticals has not been completely solved. To eliminate all plant-specific N-glycan structure, eight genes involved in plant-specific N-glycosylation were mutated in rice (Oryza sativa) using the CRISPR/Cas9 system. The glycoengineered cell lines, PhytoRice®, contained a predominant GnGn (G0) glycoform. The gene for codon-optimized trastuzumab (TMab) was then introduced into PhytoRice® through Agrobacterium co-cultivation. Selected cell lines were suspension cultured, and TMab secreted from cells was purified from the cultured media. The amino acid sequence of the TMab produced by PhytoRice® (P-TMab) was identical to that of TMab. The inhibitory effect of P-TMab on the proliferation of the BT-474 cancer cell line was significantly enhanced at concentrations above 1 µg/mL (****P < 0.0001). P-TMab bound to a FcγRIIIa variant, FcγRIIIa-F158, more than 2.7 times more effectively than TMab. The ADCC efficacy of P-TMab against Jurkat cells was 2.6 times higher than that of TMab in an in vitro ADCC assay. Furthermore, P-TMab demonstrated efficient tumour uptake with less liver uptake compared to TMab in a xenograft assay using the BT-474 mouse model. These results suggest that the glycoengineered PhytoRice® could be an alternative platform for mAb production compared to current CHO cells, and P-TMab has a novel and enhanced efficacy compared to TMab.

ß1,3-galactosyltransferase on chromosome 6 is essential for the formation of Lewis a structure on N-glycan in Oryza sativa.

ß1,3-galactose is the component of outer-chain elongation of complex N-glycans that, together with α1,4-fucose, forms Lewis a structures in plants. Previous studies have revealed that N-glycan maturation is mediated by sequential attachment of ß1,3-galactose and α1,4-fucose by individual ß1,3-galactosyltransferase (GalT) and α1,4-fucosyltransferase (1,4-FucT), respectively. Although GalT from several species has been studied, little information about GalT from rice is available. I therefore characterized three GalT candidate genes on different chromosomes in Oryza sativa. Seeds of rice lines that had T-DNA insertions in regions corresponding to individual putative GalT genes were obtained from a Rice Functional Genomic Express Database and plants grown until maturity. Homozygotes were selected from the next generation by genotyping PCR, and used for callus induction. Callus extracts of two independent T-DNA mutant rice which have T-DNA insertions at the same gene on chromosome 6 but in different exons showed highly reduced band intensity on a western blots using an anti-Lewis a antibody. Cell extracts and cultured media from suspension culture of the one of these mutant rice were further analysed by N-glycan profiling using matrix-associated laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF). Identified N-glycan species containing ß1,3-galactose from both cell extracts and cultured media of knock-out mutant were less than 0.5% of total N-glycans while that of WT cells were 9.8% and 49.1%, respectively. This suggests that GalT located on rice chromosome 6 plays a major role in N-glycan galactosylation, and mutations within it lead to blockage of Lewis a epitope formation.

Production of Plant-Derived Japanese Encephalitis Virus Multi-Epitope Peptide in Nicotiana benthamiana and Immunological Response in Mice.

The current production of the Japanese encephalitis virus (JEV) vaccine is based on animal cells, where various risk factors for human health should be resolved. This study used a transient expression system to express the chimeric protein composed of antigenic epitopes from the JEV envelope (E) protein in Nicotiana benthamiana. JEV multi-epitope peptide (MEP) sequences fused with FLAG-tag or 6× His-tag at the C- or N-terminus for the purification were introduced into plant expression vectors and used for transient expression. Among the constructs, vector pSK480, which expresses MEP fused with a FLAG-tag at the C-terminus, showed the highest level of expression and yield in purification. Optimization of transient expression procedures further improved the target protein yield. The purified MEP protein was applied to an ICR mouse and successfully induced an antibody against JEV, which demonstrates the potential of the plant-produced JEV MEP as an alternative vaccine candidate.

Comparison of oncologic outcomes between open and laparoscopic distal pancreatectomy for pancreatic ductal adenocarcinoma using data from the KOTUS-BP national database: overcoming selection bias and the necessity of definite indications.

BACKGROUND: Despite the lack of high-level evidence, laparoscopic distal pancreatectomy (LDP) is frequently performed in patients with pancreatic ductal adenocarcinoma (PDAC) owing to advancements in surgical techniques. The aim of this study was to investigate the long-term oncologic outcomes of LDP in patients with PDAC via propensity score matching (PSM) analysis using data from a large-scale national database. METHODS: A total of 1202 patients who were treated for PDAC via distal pancreatectomy across 16 hospitals were included in the Korean Tumor Registry System-Biliary Pancreas. The 5-year overall (5YOSR) and disease-free (5YDFSR) survival rates were compared between LDP and open DP (ODP). RESULTS: ODP and LDP were performed in 846 and 356 patients, respectively. The ODP group included more aggressive surgeries with higher pathologic stage, R0 resection rate, and number of retrieved lymph nodes. After PSM, the 5YOSRs for ODP and LDP were 37.3% and 41.4% (p = 0.150), while the 5YDFSRs were 23.4% and 27.2% (p = 0.332), respectively. Prognostic factors for 5YOSR included R status, T stage, N stage, differentiation, and lymphovascular invasion. CONCLUSION: LDP was performed in a selected group of patients with PDAC. Within this group, long-term oncologic outcomes were comparable to those observed following ODP.

Inactivation of the ß (1, 2)-xylosyltransferase and the α (1, 3)-fucosyltransferase gene in rice (Oryza sativa) by multiplex CRISPR/Cas9 strategy.

KEY MESSAGE: CRISPR/Cas9-mediated OsXylT and OsFucT mutation caused the elimination of plant-specific ß1,2-xylose and α1,3-fucose residues on glycoproteins in rice, which is the first report of OsXylT/OsFucT double KO mutation in rice. N-glycosylation pathway is the one of post-translational mechanism and is known as highly conserved in eukaryotes. However, the process for complex-N-glycan modification is different between mammals and plants. In plant-specific manner, ß1,2-xylose and α1,3-fucose residues are transferred to N-glycan core structure on glycoproteins by ß1,2-xylosyltransferase (ß1,2-XylT) and α1,3-fucosyltransferase (α1,3-FucT), respectively. As an effort to use plants as a platform to produce biopharmaceuticals, the plant-specific N-glycan genes of rice (Oryza sativa), ß1,2-xylT (OsXylT) and α1,3-FucT (OsFucT), were knocked out using multiplex CRISPR/Cas9 technology. The double knock-out lines were found to have frameshift mutations by INDELs. Both ß1,2-xylose and α1,3-fucose residues in the lines were not detected in Western blot analysis. Consistently, there was no peak corresponding to the N-glycans in MALDI-TOF/MS analysis. Although α1,3-fucose and ß1,2-xylose residues were not detected in the line, other plant-specific residues of ß1,3-galactose and α1,4-fucose were detected. Thus, we suggest that each enzymes working on the process for complex N-glycan biosynthesis might independently act in rice, hence the double knock-out of both OsXylT and OsFucT might be not enough to humanize N-glycan structure in rice.

OsCBE1, a Substrate Receptor of Cullin4-Based E3 Ubiquitin Ligase, Functions as a Regulator of Abiotic Stress Response and Productivity in Rice.

Ubiquitination is an important environmental stress response, and E3 ubiquitin ligases play a major role in the process. T-DNA insertion mutants of rice, Oscbe1-1, and Oscbe1-2, were identified through the screening of cold stress tolerance at seedling stage. Oscbe1 mutants showed a significantly higher cold stress tolerance in the fresh weight, chlorophyll content, and photosynthetic efficiency than wild type. Molecular prediction showed that OsCBE1 (Oryza sativa Cullin4-Based E3 ubiquitin ligase1) encoded a novel substrate receptor of Cullin4-based E3 ubiquitin ligase complex (C4E3). Whereas Oscbe1 mutants had fewer panicles and grains than wild type in the paddy field, the overexpression lines of OsCBE1 had more panicles and grains, suggesting that OsCBE1 is involved in the regulation of both abiotic stress response and development. Oscbe1 mutants also showed ABA hypersensitivity during seed germination, suggesting OsCBE1 function for the stress response via ABA signaling. In silico analysis of OsCBE1 activity predicted a CCCH-type transcription factor, OsC3H32, as a putative substrate. Co-IP (Co-immunoprecipitation) study showed that OsCBE1 interacts with OsDDB1, an expected binding component of OsCBE1 and OsC3H32. Additionally, expression of OsOLE16, OsOLE18, and OsBURP5 were negatively related with expression of OsCBE1. These results suggest that OsCBE1 functions as a regulator of the abiotic stress response via CCCH as a member of the C4E3.

Systematic Analysis of Cold Stress Response and Diurnal Rhythm Using Transcriptome Data in Rice Reveals the Molecular Networks Related to Various Biological Processes.

Rice (Oryza sativa L.), a staple crop plant that is a major source of calories for approximately 50% of the human population, exhibits various physiological responses against temperature stress. These responses are known mechanisms of flexible adaptation through crosstalk with the intrinsic circadian clock. However, the molecular regulatory network underlining this crosstalk remains poorly understood. Therefore, we performed systematic transcriptome data analyses to identify the genes involved in both cold stress responses and diurnal rhythmic patterns. Here, we first identified cold-regulated genes and then identified diurnal rhythmic genes from those (119 cold-upregulated and 346 cold-downregulated genes). We defined cold-responsive diurnal rhythmic genes as CD genes. We further analyzed the functional features of these CD genes through Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses and performed a literature search to identify functionally characterized CD genes. Subsequently, we found that light-harvesting complex proteins involved in photosynthesis strongly associate with the crosstalk. Furthermore, we constructed a protein-protein interaction network encompassing four hub genes and analyzed the roles of the Stay-Green (SGR) gene in regulating crosstalk with sgr mutants. We predict that these findings will provide new insights in understanding the environmental stress response of crop plants against climate change.

Lack of the α1,3-Fucosyltransferase Gene (Osfuct) Affects Anther Development and Pollen Viability in Rice.

N-linked glycosylation is one of the key post-translational modifications. α1,3-Fucosyltransferase (OsFucT) is responsible for transferring α1,3-linked fucose residues to the glycoprotein N-glycan in plants. We characterized an Osfuct mutant that displayed pleiotropic developmental defects, such as impaired anther and pollen development, diminished growth, shorter plant height, fewer tillers, and shorter panicle length and internodes under field conditions. In addition, the anthers were curved, the pollen grains were shriveled, and pollen viability and pollen number per anther decreased dramatically in the mutant. Matrix-assisted laser desorption/ionization time-of-flight analyses of the N-glycans revealed that α1,3-fucose was lacking in the N-glycan structure of the mutant. Mutant complementation revealed that the phenotype was caused by loss of Osfuct function. Transcriptome profiling also showed that several genes essential for plant developmental processes were significantly altered in the mutant, including protein kinases, transcription factors, genes involved in metabolism, genes related to protein synthesis, and hypothetical proteins. Moreover, the mutant exhibited sensitivity to an increased concentration of salt. This study facilitates a further understanding of the function of genes mediating N-glycan modification and anther and pollen development in rice.

Totally laparoscopic or robot-assisted pancreaticoduodenectomy versus open surgery for periampullary neoplasms: separate systematic reviews and meta-analyses.

OBJECTIVE: To compare perioperative and oncologic outcomes of pure (totally) laparoscopic pancreaticoduodenectomy (TLPD) or robot-assisted pancreaticoduodenectomy (RAPD) with those of conventional open pancreaticoduodenectomy (OPD). METHODS: A systematic literature search was performed using PubMed, EMBASE, and Cochrane library databases. Studies comparing TLPD with OPD and RAPD with OPD were included; only original studies reporting more than 10 cases for each technique were included. Studies were combined using a random-effects model to report heterogeneous data, or a fixed-effects model was applied. RESULTS: TLPD involved longer operative time (weighted mean difference [WMD]: 116.85 min; 95% confidence interval [CI] 54.53-179.17) and significantly shorter postoperative hospital stay (WMD: -3.68 days; 95% CI -4.65 to -2.71). Overall morbidity and postoperative pancreatic fistula were not significantly different between TLPD and OPD. RAPD was associated with a longer operative time, less intraoperative blood loss, and shorter hospital stay. Oncologic outcomes were not significantly different among the procedure types. CONCLUSIONS: Compared to OPD, TLPD and RAPD were feasible and oncologically safe procedures. However, there are no prospective studies, and the majority of the studies on TLPD and RAPD have remained in the early training phase. In addition to randomized controlled trials or prospective studies, new data from the late training phase of learning experiences should also be analyzed.

A single-center experience with the laparoscopic Warshaw technique in 122 consecutive patients.

BACKGROUND: Preservation of the spleen in distal pancreatectomy has recently attracted considerable attention. Our current study aimed in the first instance to define the safety of lap-WT in relation to the capacity of this technique to achieve preservation of the spleen and secondly to investigate the effectiveness of a planned lap-WT procedure or early conversion to lap-WT in selected patients with a large tumor attached to the splenic vessels. METHODS: Among 1056 patients who underwent a laparoscopic distal pancreatectomy between January 2005 and December 2014 at our hospital, 122 (24.6 %) underwent lap-WT which were analyzed. The 122 patients were categorized into two groups chronologically (early group: 2005-2012, late group: 2013-2014). RESULTS: The median follow-up was 35 months, and the median operation time was 181 min. The median postoperative hospital stay was 7 days, and the median estimated blood loss was 316 ml. Postoperative complications occurred in 9 patients (7.3 %), including 4 patients (3.2 %) with major pancreatic fistula (ISGPF grade B, C). A reoperation to address postoperative bleeding was needed in one patient. During a median follow-up of 35 months, there were no clinical significant splenic infarctions or gastric varices in any case. All patients were observed conservatively. In patients in the late group who underwent the lap-WT, the mean operating time (171 vs. 205 min, p = 0.001) and mean estimated blood loss (232.1 vs. 370.0 ml, p = 0.017) were significantly less than the early group cases who received lap-WT. CONCLUSIONS: A lap-WT is a safe treatment strategy in select cases when used as a way of preserving the spleen. When splenic vessel preservation is technically challenging, for example when the tumor is enlarged or is attached to the splenic vessels, planned lap-WT or early conversion to lap-WT may be a feasible option.

Robotic liver resection: a single surgeon's experience.

BACKGROUND: Liver resection has long been a complicated challenge in terms of minimally invasive surgery. However, robotic surgery has expanded the number of surgical procedures that can be performed using minimally invasive techniques. This study describes the authors' experience of 17 robotic liver resections performed using the da Vinci Surgical System. METHODS: From May 2010 to May 2012, 17 patients underwent robotic liver resection at Asan Medical Center, Seoul, Korea. Only patients who underwent left hepatectomy or left lateral sectionectomy were included in the study. RESULTS: Thirteen patients had hepatocellular carcinoma, one had a biliary cyst, one had a dysplastic nodule, one had fibronodular hyperplasia, and one had a left intrahepatic duct stone. The mean operative time was 267.06 ± 84.62 minutes and the mean estimated blood loss was 264.71 ± 104.23 mL. No open conversion was required. The mean tumor size was 2.98 ± 1.47 cm and the mean hospital stay was 7.58 ± 2.26 days. CONCLUSION: The results confirm the safety and feasibility of robotic liver resection. As surgeons become more experienced with robotic liver resection and the technology improves, more patients will benefit from this approach.

Clinical implication of the geometric location (fundal end versus cystic ductal end) of gallbladder cancer.

BACKGROUND: Management of early-stage gallbladder cancer is becoming more important as the rate of early detection is increasing. Although there have been many studies about the clinical implication of the invasion depth or peritoneal/hepatic location of gallbladder cancers, there is no study on the clinical implication of the geometric location of cancer along the longitudinal length of the gallbladder. METHODS: The location of gallbladder cancer was defined as the geometric center of the primary site of a tumour, which lies on the longitudinal diameter of the surgical specimens. We compared the oncologic outcomes following surgery between gallbladder cancers located on the fundal end and those located on the cystic ductal end. We also analysed patients with stage 1 gallbladder cancer who recurred after surgery. RESULTS: A total of 575 patients with gallbladder cancer were included in this study. Patients with gallbladder cancer on the cystic ductal end had significantly lower rates of recurrence-free survival (P = 0.016) and overall survival (P = 0.023) compared to those with gallbladder cancer on the fundal end. Among 90 patients with stage 1 gallbladder cancer, three patients had a recurrence, all of whom had cystic ductal end gallbladder cancer and showed cystic duct invasion or concomitant xanthogranulomatous cholecystitis in permanent pathology. CONCLUSIONS: Gallbladder cancers on the cystic ductal end had worse postoperative oncologic outcomes compared with those on the fundal end.

Consistent Response on Challenge and Rechallenge of Liposomal Irinotecan in a Patient with Metastatic Pancreatic Adenocarcinoma Previously Treated with Gemcitabine plus Nab-Paclitaxel: A Case Report.

Approximately 80% of pancreatic cancer is diagnosed at an advanced stage, due to lack of or vague symptoms when the cancer is still localized, leading to a high mortality rate. Known risk factors for developing pancreatic cancer are family history, obesity, type 2 diabetes, and alcohol and tobacco use. There has been a remarkable development in diagnosis modalities and molecular testing, but early detection is still infrequent. The majority of clinical trials have not shown significant efficacy in pancreatic cancer, and treatment strategy remains limited. Additional prognostic factors should be highlighted to obtain appropriate treatment options, including precision medicine, and improve survival outcomes. After the PRODIGE study in 2011 and the MPAC trial in 2013, a new drug (liposomal irinotecan; Onivyde ®) appeared in the strategy, especially after failure of gemcitabine-based treatment. In 2016, the NAPOLI-1 trial showed evidence of the efficacy of the liposomal irinotecan combination (liposomal irinotecan +5-fluorouracile + folinic acid); now, it is considered the standard treatment for relapsing patients. Since NAPOLI-1, real-world data have provided similar results. Herein, we report the story of a 61-year-old woman who was treated with liposomal irinotecan combination (nal-IRI/5-FU/LV) for 8 months with good surgical response, but treatment was discontinued due to economic burden. After the start of treatment (or 1? cycle of liposomal irinotecan treatment), the patient was in a better condition. The liver metastases had disappeared. The combination with liposomal irinotecan was re-administered with patient's approval. Upon rechallenge with the liposomal irinotecan combination, she showed a partial response, and the treatment was given for 7 months. In this report, we tried to identify the prognostic factors leading to the efficacy of the liposomal irinotecan combination.

Surgical outcomes are hampered after endoscopic ultrasonography-guided ethanol lavage and/or Taxol injection in cystic lesions of the pancreas.

BACKGROUNDS/AIMS: Endoscopic ultrasonography-guided ethanol lavage and Taxol injection (EUS-ELTI) for pancreatic cystic lesions have been recently performed in some medical centers. The aim of this study was to optimize patient selection and analyze outcomes of patients who underwent surgeries after EUS-ELTI for pancreatic cystic lesions. METHODS: Among 310 patients who underwent EUS-ELTI between January 2007 and December 2014, 23 underwent surgeries after EUS-ELTI owing to incomplete treatment and/or adverse events. Surgical outcomes of patients who underwent surgeries after EUSELTI were evaluated. Clinical outcomes of patients who underwent surgeries after EUS-ELTI were then retrospectively compared with those of patients who underwent upfront surgery for left-sided pancreatic lesions without an EUS-ELTI procedure. RESULTS: The pathology revealed degenerated cysts in 12 patients, mucinous cyst neoplasms in five, neuroendocrine tumors in two, intraductal papillary mucinous neoplasm (IPMN) in one, solid pseudopapillary tumor in one, pancreatic ductal adenocarcinoma arising from an IPMN in one, and hepatoid carcinoma in one. Twelve patients underwent laparoscopic distal pancreatectomy and five patients underwent open distal pancreatectomy. When clinical outcomes were retrospectively compared between patients who underwent laparoscopic distal pancreatectomy after EUS-ELTI and those who did not receive an EUS-ELTI procedure, the spleen-preserving rate was 0% in the EUS-ELTI group and 61.7% (365/592) in the control group (p < 0.001). CONCLUSIONS: Surgical outcomes are compromised after EUS-ELTI for cystic tumor of the pancreas. Further studies are needed to investigate the efficacy and safety of the EUS-ELTI procedure.

Development and External Validation of Survival Prediction Model for Pancreatic Cancer Using Two Nationwide Databases: Surveillance, Epidemiology and End Results (SEER) and Korea Tumor Registry System-Biliary Pancreas (KOTUS-BP).

Background/Aims: Several prediction models for evaluating the prognosis of nonmetastatic resected pancreatic ductal adenocarcinoma (PDAC) have been developed, and their performances were reported to be superior to that of the 8th edition of the American Joint Committee on Cancer (AJCC) staging system. We developed a prediction model to evaluate the prognosis of resected PDAC and externally validated it with data from a nationwide Korean database. Methods: Data from the Surveillance, Epidemiology and End Results (SEER) database were utilized for model development, and data from the Korea Tumor Registry System-Biliary Pancreas (KOTUS-BP) database were used for external validation. Potential candidate variables for model development were age, sex, histologic differentiation, tumor location, adjuvant chemotherapy, and the AJCC 8th staging system T and N stages. For external validation, the concordance index (C-index) and time-dependent area under the receiver operating characteristic curve (AUC) were evaluated. Results: Between 2004 and 2016, data from 9,624 patients were utilized for model development, and data from 3,282 patients were used for external validation. In the multivariate Cox proportional hazard model, age, sex, tumor location, T and N stages, histologic differentiation, and adjuvant chemotherapy were independent prognostic factors for resected PDAC. After an exhaustive search and 10-fold cross validation, the best model was finally developed, which included all prognostic variables. The C-index, 1-year, 2-year, 3-year, and 5-year time-dependent AUCs were 0.628, 0.650, 0.665, 0.675, and 0.686, respectively. Conclusions: The survival prediction model for resected PDAC could provide quantitative survival probabilities with reliable performance. External validation studies with other nationwide databases are needed to evaluate the performance of this model.

The establishment of new protein expression system using N starvation inducible promoters in Chlorella.

Chlorella is a unicellular green microalga that has been used in fields such as bioenergy production and food supplementation. In this study, two promoters of N (nitrogen) deficiency-inducible Chlorella vulgaris N Deficiency Inducible (CvNDI) genes were isolated from Chlorella vulgaris UTEX 395. These promoters were used for the production of a recombinant protein, human granulocyte-colony stimulating factor (hG-CSF) in Chlorella vulgaris UTEX 395 and Chlorella sp. ArM0029B. To efficiently secrete the hG-CSF, the protein expression vectors incorporated novel signal peptides obtained from a secretomics analysis of Chlorella spp. After a stable transformation of those vectors with a codon-optimized hG-CSF sequence, hG-CSF polypeptides were successfully produced in the spent media of the transgenic Chlorella. To our knowledge, this is the first report of recombinant protein expression using endogenous gene components of Chlorella.

Multifrequency Bioelectrical Impedance Analysis Compared With Computed Tomography for Assessment of Skeletal Muscle Mass in Primary Colorectal Malignancy: A Predictor of Short-Term Outcome After Surgery.

BACKGROUND: Herein, we evaluate the accuracy and reliability of multifrequency bioelectrical impedance analysis (BIA) to assess skeletal muscle mass (SMM) in the colorectal cancer patient compared with computed tomography (CT) scan and also analyze the association between SMM of BIA and postoperative outcomes. METHODS: From March to May 2017, the body composition of patients scheduled for elective surgery due to primary colorectal cancer in our institution was analyzed at the time of admission using BIA (InBody S20, Biospace Co Ltd, Seoul, South Korea). The relationship between the single cross-sectional area of skeletal muscle at the lumbar region using preoperative CT scan and the SMM determined via BIA were assessed. Postoperative outcomes were compared according to different status of SMM on BIA categorized into 3 groups (low, medium, or high). RESULTS: Fifty patients were analyzed, and the length of hospital stay was shorter and the initiation of oral diet was significantly earlier in the group with high SMM than in other groups (P = 0.001 and 0.038, respectively). The SMM on BIA showed a very significant correlation with skeletal muscle index using CT scan (0.705 of correlation coefficients, P < 0.001) and also strongly correlated with skeletal muscle index after adjusting for age, weight, and BMI on multivariate analysis (ß = 0.391 ± 0.057, P < 0.001). CONCLUSION: SMM determined by BIA is strongly correlated with SMM estimated by CT scan. Authors suppose that BIA could be an alternative to CT scan for the assessment of SMM in colorectal cancer patients.

Heat-inducible C3HC4 type RING zinc finger protein gene from Capsicum annuum enhances growth of transgenic tobacco.

Capsicum annuum RING Zinc Finger Protein 1 (CaRZFP1) gene is a novel C3HC4-type RING zinc finger protein gene which was previously isolated from a cDNA library for hot pepper plants treated of heat-shock. The CaRZFP1 was inducible to diverse environmental stresses in hot pepper plants. We introduced the CaRZFP1 into the Wisconsin 38 cultivar of tobacco (Nicotiana tabacum) by Agrobacterium mediated transformation under the control of the CaMV 35S promoter. Expression of the transgene in the transformed tobacco plants was demonstrated by RNA blot analyses. There appeared no adverse effect of over-expression of the transgene on overall growth and development of transformants. The genetic analysis of tested T(1) lines showed that the transgene segregated in a Mendelian fashion. Transgenic tobacco lines that expressed the CaRZFP1 gene were compared with several different empty vector lines and they exhibited enhanced growth; they have larger primary root, more lateral root, larger hypocotyls and bigger leaf size, resulting in heavier fresh weight. Enhanced growth of transgenic lines accompanied with longer vegetative growth that resulted in bigger plants with higher number of leaves. Microarray analysis revealed the up-regulation of some growth related genes in the transgenic plants which were verified by specific oligomer RNA blot analyses. These results indicate that CaRZFP1 activates and up-regulates some growth related proteins and thereby effectively promoting plant growth.

Engineered mesenchymal stem-cell-sheets patches prevents postoperative pancreatic leakage in a rat model.

Post-operative pancreatic fistula (POPF) following pancreatic resection is a life-threatening surgical complication. Cell sheets were prepared and harvested using temperature-responsive culture dishes and transplanted as patches to seal POPF. Two different mesenchymal stem cell (MSC) sheets were compared in terms of the preventative ability for pancreatic leakage in a rat model. Both rat adipose-derived stem cell (rADSC) and bone marrow-derived stem cell (rBMSC) sheets were transplanted. Those rADSC and rBMSC sheets are created without enzymes and thus maintained their cell-cell junctions and adhesion proteins with intact fibronectin on the basal side, as well as characteristics of MSCs. The rats with post-pancreatectomy rADSC- or rBMSC-sheet patches had significantly decreased abdominal fluid leakage compared with the control group, demonstrated by MR image analysis and measurement of the volume of abdominal fluid. Amylase level was significantly lower in the rats with rADSC-sheet and rBMSC-sheet patches compared with the control groups. The rADSC sheet patches had increased adhesive and immune-cytokine profiles (ICAM-1, L-selectin, TIMP-1), and the rBMSC sheets had reduced immune reactions compared to the control. This is first project looking at the feasibility of tissue engineering therapy using MSC-sheets as tissue patches preventing leakage of abdominal fluid caused by POPF.

Ectopic Expression of OsSta2 Enhances Salt Stress Tolerance in Rice.

Salt stress can severely reduce crop yields. To understand how rice (Oryza sativa) plants respond to this environmental challenge, we investigated the genes involved in conferring salt tolerance by screening T-DNA tagging lines and identified OsSta2-D (Oryza sativa Salt tolerance activation 2-Dominant). In that line, expression of OsSta2 was enhanced by approximately eightfold when compared with the non-transformed wild type (WT). This gene was highly expressed in the callus, roots, and panicles. To confirm its role in stress tolerance, we generated transgenic rice that over-expresses OsSta2 under a maize ubiquitin promoter. The OsSta2-Ox plants were salt-tolerant at the vegetative stage, based on our calculations of chlorophyll fluorescence (Fv/Fm), fresh and dry weights, chlorophyll concentrations, and survival rates. Under normal paddy field conditions, the Ox plants were somewhat shorter than the WT control but had improved agronomic traits such as higher total grain yield. They were also more tolerant to osmotic stress and hypersensitive to abscisic acid. Based on all of these results, we suggest that OsSta2 has important roles in determining yields as well as in conferring tolerance to salt stresses.