Critical Hemorrhage Caused by a Size-Mismatched Extracorporeal Membrane Oxygenation Cannula in a Patient with Myotonic Dystrophy Type 1: A Case Report and Literature Review.

Background and Objective: Although extracorporeal membrane oxygenation (ECMO) is an essential life-saving technique for patients with refractory cardiopulmonary shock, it can be fatal in certain cases. Case Presentation: A 19-year-old girl treated with ECMO presented with acute limb ischemia 2 days after cannula removal. The decannulation was performed percutaneously by an interventional cardiologist, and the vascular surgery department was consulted after the patient developed symptoms. The first suspected diagnosis was thrombosis due to incorrect use of the closure device. However, the artery had ruptured due to the insertion of a catheter with a cannula that was larger than the patient's artery. Management and Outcome: Fortunately, excessive bleeding due to the size-mismatched cannula was prevented by an unintentional complication of the closing device, which saved the patient's life. She underwent a right common femoral artery thrombectomy and patch angioplasty. Hospital guidelines have changed regarding the surgical removal of ECMO cannulas. Discussion: This report aims to highlight the importance of two aspects that are critical to a successful outcome: individualized cannula selection followed by precise insertion and removal and postoperative evaluation of a patient's final status.

Rupture of Mycotic Abdominal Aortic Aneurysm as a Result of Incompletely Treated Multiple Peripheral Mycotic Aneurysms.

Background: A mycotic aortic aneurysm is a rare type of aortic aneurysm that can have disastrous outcomes. Most mycotic aneurysms originate from infectious sources, such as trauma, vegetation in the heart, and adjacent infectious sources. If a mycotic aneurysm is diagnosed, it should be treated simultaneously with the primary source of the infection. Case Summary: Treatment was performed for a mycotic aneurysm of the brachial artery that occurred suddenly during treatment for a fever for which the primary source of infection had not been confirmed. The workup revealed that a mycotic aneurysm of the brachial artery was the cause of the fever, followed by aneurysms in the abdomen and lower extremities and even vegetation in the heart that was not initially present. The patient declined to undergo treatment for personal reasons. After 5 months, it was revealed that the abdominal aortic aneurysm, which was initially considered normal aorta, was ruptured; however, the aneurysm was successfully treated. Conclusions: A peripheral mycotic aneurysm may be associated with multiple aneurysms. Appropriate diagnosis and complete treatments are necessary to prevent fatal consequences.

An Asymptomatic Sigmoid Colonic Fistula Arising from a Large Aneurysm of the Internal Iliac Artery Was Discovered during a Medical Examination.

The rupture of an internal iliac artery aneurysm in the colon is a rare but potentially fatal complication. We report a rectal fistula of an asymptomatic internal iliac artery aneurysm that was discovered incidentally during a medical examination. A 77-year-old man presented at a local hospital for a general medical examination. Although the blood reports revealed severe anemia, the patient did not complain of any associated symptoms including dizziness and hematochezia. Moreover, there was no palpable mass in the patient's abdomen, and there was no evidence of hematochezia, as the patient had been using a bidet. Interestingly, computed tomography (CT) revealed a large right internal iliac artery aneurysm. There was a suspicious finding of a fistula within the colon in the CT, but it was undetected in the preoperative sigmoidoscopy. Furthermore, operative findings showed a protruding retroperitoneal mass adhering to the mesentery of the sigmoid colon. During aneurysm resection, the presence of a fistula was unclear. However, a fistula tract, devoid of any infectious bacteria such as tuberculosis, was found in the specimen after colon resection. After a recovery period of approximately one week, the patient was discharged from the hospital without any unusual findings on the post-operative CT. Sigmoid colonic fistulas arising from iliac artery aneurysms are rare. Also, diagnosis may be delayed in special circumstances wherein a patient routinely uses a bidet.

Gastric Metastasis Mimicking Early Gastric Cancer from Invasive Ductal Carcinoma of the Breast: Case Report and Literature Review.

Backgound and Objectives: Gastric metastasis from invasive ductal breast cancer (BC) is rare. It mainly occurs in patients with lobular BC. The occurrence of multiple metastases is typically observed several years after the primary diagnosis. Endoscopic findings of gastric metastasis of the BC were usually the linitis plastic type. Case presentation: A 72-year-old women who underwent right modified radical mastectomy (MRM) 10 month ago was referred after being diagnosed with early gastric cancer (EGC) during systemic chemotherapy. EGC type I was found at gastric fundus, and pathologic finding showed poorly differentiated adenocarcinoma. Metachronous double primary tumor EGC was considered. Management and Outcome: A laparoscopic total gastrectomy was performed, and postoperative pathology revealed submucosa invasion and two lymph node metastases. A pathologic review that focused on immunohistochemical studies of selected antibodies such as GATA binding protein 3 (GATA3), gross cystic disease fluid protein-15 (GCDFP-15), cytokeratin 7 (CK7) was performed again, comparing previous results. As a result, gastric metastasis from BC was diagnosed. After totally laparoscopic total gastrectomy, palliative first-line chemotherapy with paclitaxel/CDDP was performed. Two months after gastrectomy, she was diagnosed with para-aortic lymph node metastasis and multiple bone metastases. She expired six months after gastrectomy. Conclusions: Gastric metastasis from invasive ductal carcinoma of the breast, which is clinically manifested as EGC, is a very rare condition. If there is a history of BC, careful pathological review will be required.

Vasomotion in human arteries and their regulations based on ion channel regulations: 10 years study.

Vasomotion is the oscillation of vascular tone which gives rise to flow motion of blood into an organ. As is well known, spontaneous contractile organs such as heart, GI, and genitourinary tract produce rhythmic contraction. It imposes or removes pressure on their vessels alternatively for exchange of many substances. It was first described over 150 years ago, however the physiological mechanism and pathophysiological implications are not well understood. This study aimed to elucidate underlying mechanisms and physiological function of vasomotion in human arteries. Conventional contractile force measurement, immunohistochemistry, and Western blot analysis were employed to study human left gastric artery (HLGA) and uterine arteries (HUA). RESULTS: Circular muscle of HLGA and/or HUA produced sustained tonic contraction by high K+ (50 mM) which was blocked by 2 µM nifedipine. Stepwise stretch and high K+ produced nerve-independent spontaneous contraction (vasomotion) (around 45% of tested tissues). Vasomotion was also produced by application of BayK 8644, 5-HT, prostagrandins, oxytocin. It was blocked by nifedipine (2 µM) and blockers of intracellular Ca2+ stores. Inhibitors of Ca2+ -activated Cl- channels (DIDS and/or niflumic acid) and ATP-sensitive K+ (KATP ) channels inhibited vasomotion reversibly. Metabolic inhibition by sodium cyanide (NaCN) and several neuropeptides also regulated vasomotion in KATP channel-sensitive and -insensitive manner. Finally, we identified TMEM16A Ca2+ -activated Cl- channels and subunits of KATP channels (Kir 6.1/6.2 and sulfonylurea receptor 2B [SUR2B]), and c-Kit positivity by Western blot analysis. We conclude that vasomotion is sensitive to TMEM16A Ca2+ -activated Cl- channels and metabolic changes in human gastric and uterine arteries. Vasomotion might play an important role in the regulation of microcirculation dynamics even in pacemaker-related autonomic contractile organs in humans.

Plk2-mediated phosphorylation and translocalization of Nrf2 activates anti-inflammation through p53/Plk2/p21cip1 signaling in acute kidney injury.

The Plk2 is a cellular stress-responsive factor that is induced in response to oxidative stress. However, the roles of Plk2 in acute kidney injury (AKI) have not been clarified. We previously found that Plk2 is an interacting factor of Nrf2 in response to cellular stress, since Plk2 is upregulated in the Nrf2-dependent network. Here, we show that the levels of p53, Plk2, p21cip1, and chromatin-bound Nrf2 were all upregulated in kidney tissues of mice or NRK52E cells treated with either cisplatin or methotrexate. Upregulation of Plk2 by p53 led to an increase of Nrf2 in both soluble and chromatin fractions in cisplatin-treated NRK52E cells. Consistently, depletion of Plk2 suppressed the levels of Nrf2. Of note, Plk2 directly phosphorylated Nrf2 at Ser40, which facilitated its interaction with p21cip1 and translocation into the nuclei for the activation of anti-oxidative and anti-inflammatory factors in response to AKI. Together, these findings suggest that Plk2 may serve as an anti-oxidative and anti-inflammatory regulator through the phosphorylation and activation of Nrf2 to protect kidney cells from kidney toxicants and that Plk2 and Nrf2 therefore work cooperatively for the protection and survival of kidney cells from harmful stresses.

Craniofacial and dental injuries associated with stand-up electric scooters.

BACKGROUND/AIM: With the recent increased share of stand-up electric scooters (e-scooters), it is common to see people riding e-scooters on the roads in Korea. The aim of this study was to investigate traumatic injuries to the craniofacial region related to e-scooter accidents and to determine the role of dentists (especially oral and maxillofacial surgeons) in the evaluation of patients with trauma at the emergency department due to an e-scooter accident. MATERIALS AND METHODS: This retrospective study investigated the medical records of patients who visited the Gangnam Severance Hospital Emergency Care Center for trauma related to e-scooter use from January 1, 2017 to March 31, 2020. Medical records were reviewed to determine the injuries sustained to the craniofacial region related to e-scooter use, including location of the injury (eg, cranium, craniofacial bone, teeth, soft tissue) and the type of trauma (eg, fracture, laceration, abrasion, contusion, concussion). RESULT: A total of 256 patients' medical records were evaluated. Among them, 125 patients (48.8% of all patients) had sustained craniofacial trauma. Laceration (n = 56, 44.8%) was the most common type of craniofacial injury, followed by cerebral concussion (n = 49, 39.2%), dental injury (n = 27, 21.6%), and craniofacial bone fracture (n = 16, 12.8%). CONCLUSION: Dentists should always consider the possibility of brain trauma and perform a complete craniofacial and oral examination when assessing patients after e-scooter accidents as outlined by the International Association of Dental Traumatology guidelines. Additionally, it is necessary to educate e-scooter riders about the importance of wearing protective devices, such as helmets, to reduce the risk of injuries to the craniofacial region.

The establishment of a rheumatoid arthritis primate model in Macaca fascicularis.

BACKGROUND: Rheumatoid arthritis (RA) is a long-term autoimmune disorder that mostly affects the joints and leads to the destruction of cartilage. An RA model in non-human primates is especially useful because of their close phylogenetic relationship to humans in terms of cross-reactivity to compounds developed using modern drug technologies. METHODS: We used a collagen-induced arthritis (CIA) model in Macaca fascicularis. CIA was induced by the immunization of chicken type II collagen. Swelling was measured as the longitudinal and transverse axes of 16 proximal interphalangeal joints. RESULTS: A new system for visual evaluation was created, with a perfect score of 16. Individual behavioral analysis was also conducted. Serum was collected once a week after the first immunization. Blood chemistry and inflammatory cytokine parameters were higher in the CIA group than in the wild type group. CONCLUSION: In conclusion, we established CIA in M. fascicularis, and the results can be used for drug evaluation models.

Two-Channel Graphene pH Sensor Using Semi-Ionic Fluorinated Graphene Reference Electrode.

A reference electrode is necessary for the working of ion-sensitive field-effect transistor (ISFET)-type sensors in electrolyte solutions. The Ag/AgCl electrode is normally used as a reference electrode. However, the Ag/AgCl reference electrode limits the advantages of the ISFET sensor. In this work, we fabricated a two-channel graphene solution gate field-effect transistor (G-SGFET) to detect pH without an Ag/AgCl reference electrode in the electrolyte solution. One channel is the sensing channel for detecting the pH and the other channel is the reference channel that serves as the reference electrode. The sensing channel was oxygenated, and the reference channel was fluorinated partially. Both the channels were directly exposed to the electrolyte solution without sensing membranes or passivation layers. The transfer characteristics of the two-channel G-SGFET showed ambipolar field-effect transistor (FET) behavior (p-channel and n-channel), which is a typical characteristic curve for the graphene ISFET, and the value of VDirac was shifted by 18.2 mV/pH in the positive direction over the range of pH values from 4 to 10. The leakage current of the reference channel was 16.48 nA. We detected the real-time pH value for the two-channel G-SGFET, which operated stably for 60 min in the buffer solution.

Discordance in HER2 status between primary gastric adenocarcinoma tumors and cells from the corresponding malignant effusions.

BACKGROUND: Metastasis of gastric cancer commonly manifests as a malignant effusion, which presents an alternative cell source for human epidermal growth factor receptor 2 (HER2) status identification. This study aimed to compare HER2 status in primary gastric adenocarcinoma tumors and corresponding cell blocks prepared from malignant effusions (CB-MEs). METHODS: HER2 status was retrospectively evaluated by immunohistochemistry (IHC) in primary gastric adenocarcinomas and paired pathologically confirmed CB-MEs of 45 patients. Silver in situ hybridization (SISH) was also performed in cases with IHC 2+ for primary gastric adenocarcinomas and above IHC 1+ for CB-MEs. RESULTS: HER2 positivity was observed in 4.4% (2/45) of primary gastric adenocarcinomas and 6.7% (3/45) of CB-MEs. The HER2 concordance rate between primary gastric adenocarcinomas and CB-MEs was 88.9% (40/45) (κ = - 0.056). All five patients with HER2 positivity in the primary tumor or a CB-ME had a negative result in the corresponding paired sample. Of the 15 patients with two or more serially sampled CB-MEs, HER2 expression determined by IHC differed between each CB-ME in six (40%) patients, and all three patients with HER2 positivity in CB-MEs exhibited HER2 positivity in one of the serially sampled CB-MEs. CONCLUSIONS: The HER2 positivity rate was very low in gastric cancer patients with malignant effusions. Our results suggest that HER2 positivity was discordant between the primary gastric adenocarcinoma and corresponding CB-MEs and among serially sampled CB-MEs. The possibility of detecting HER2 positivity can be improved if the primary gastric adenocarcinoma tumor as well as all the available CB-MEs from each patient are analyzed.

Arenibacillus arenosus gen. nov., sp. nov., a member of the family Rhodobacteraceae isolated from sea sand.

A Gram-stain-negative, aerobic, non-motile, non-spore-forming, rod-shaped bacterial strain, designated CAU 1304T, was isolated from sea sand. Strain CAU 1304T grew optimally at pH 8.5 and 30 °C and in the presence of 1 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain CAU 1304T belonged to the family Rhodobacteraceae, and was most closely related to Roseicitreum antarcticum ZS2-28T (96.54 % similarity) and Rhodobacter veldkampii ATCC 35703T (96.46 %). The major fatty acid was C18 : 1ω7c and the respiratory quinone was Q-10. The polar lipids were composed of phosphatidylglycerol, phosphatidylcholine and an unidentified aminolipid. The DNA G+C content was 55.9 mol%. On the basis of phenotypic, chemotaxonomic and phylogenetic analysis, it is proposed that strain CAU 1304T represents a novel species of a new genus of the family Rhodobacteraceae, for which the name Arenibacillus arenosus gen. nov., sp. nov. is proposed. The type strain of Arenibacillus arenosus is CAU 1304T (=KCTC 42827T=NBRC 113022T).

Immediate and late outcomes of endovascular therapy for lower extremity arteries in Buerger disease.

OBJECTIVE: Buerger disease is a rare inflammatory vasculopathy presenting with severe claudication or critical limb ischemia. In this study, we sought to evaluate the feasibility and clinical outcomes of endovascular therapy for Buerger disease involving arteries in the lower extremities. METHODS: Between January 2006 and May 2016, there were 44 Buerger disease patients (43 men; mean age, 40.4 ± 9.6 years) with 50 target limbs treated by endovascular therapy at the Severance Cardiovascular Hospital. Baseline characteristics as well as both immediate and late clinical outcomes were retrospectively analyzed. RESULTS: The majority (86.4%) of patients presented with critical limb ischemia. A total of 88 target lesions in 50 limbs were treated with endovascular procedures. All limbs showed infrapopliteal artery occlusions, and multilevel diseases involving the iliac or femoropopliteal artery were found in 31 patients (62%). Technical success was achieved in 80% of subjects. We found that a lower serum level of C-reactive protein, specifically the log C-reactive protein value (odds ratio, 0.03; 95% confidence interval [CI], 0.00-0.71; P = .030), was an independent predictor of technical failure. The median follow-up duration was 29 months. Major adverse limb event-free survival and reintervention- and amputation-free survival were 83.3% and 67.9% at 3 years, respectively. In a multivariate Cox proportional hazards analysis, previous endovascular treatment (hazard ratio, 3.70; 95% CI, 1.20-11.31; P = .022) and previous amputation (hazard ratio, 4.68; 95% CI, 1.37-15.96; P = .014) were identified as independent risk factors for reintervention- and amputation-free survival. CONCLUSIONS: In patients with Buerger disease, endovascular treatment achieved technical success in the majority of the cases and was associated with favorable immediate and late clinical outcomes. These findings indicate that endovascular therapy may be considered a first-line treatment option for severe symptomatic patients with Buerger disease.

Detection of Alpha-Fetoprotein in Hepatocellular Carcinoma Patient Plasma with Graphene Field-Effect Transistor.

The detection of alpha-fetoprotein (AFP) in plasma is important in the diagnosis of hepatocellular carcinoma (HCC) in humans. We developed a biosensor to detect AFP in HCC patient plasma and in a phosphate buffer saline (PBS) solution using a graphene field-effect transistor (G-FET). The G-FET was functionalized with 1-pyrenebutyric acid N-hydroxysuccinimide ester (PBASE) for immobilization of an anti-AFP antibody. AFP was detected by assessing the shift in the voltage of the Dirac point (ΔVDirac) after binding of AFP to the anti-AFP-immobilized G-FET channel surface. This anti-AFP-immobilized G-FET biosensor was able to detect AFP at a concentration of 0.1 ng mL-1 in PBS, and the detection sensitivity was 16.91 mV. In HCC patient plasma, the biosensor was able to detect AFP at a concentration of 12.9 ng mL-1, with a detection sensitivity of 5.68 mV. The sensitivity (ΔVDirac) depended on the concentration of AFP in either PBS or HCC patient plasma. These data suggest that G-FET biosensors could have practical applications in diagnostics.

Glycyrrhizin, inhibitor of high mobility group box-1, attenuates monocrotaline-induced pulmonary hypertension and vascular remodeling in rats.

BACKGROUND: High mobility group box-1 (HMGB1), a proinflammatory cytokine, plays a pivotal role in tissue remodeling and angiogenesis, both of which are crucial for the pathogenesis of pulmonary arterial hypertension. In this study, we explored the relationship between HMGB1 and pulmonary hypertension and whether glycyrrhizin, an inhibitor of HMGB1, attenuates disease progression in an animal model of pulmonary hypertension induced by monocrotaline sodium (MCT). METHODS: After inducing pulmonary hypertension through a single subcutaneous injection of MCT (60 mg/kg) to Sprague-Dawley rats, we administered daily intraperitoneal injections of either glycyrrhizin (GLY, 50 mg/kg), an inhibitor of HMGB1, or saline (control) for either 4 or 6 weeks. RESULTS: Expression levels of HMGB1 in serum increased from the second week after MCT injection and remained elevated throughout the experiment periods. Lung tissue levels of HMGB1 assessed by immunohistochemical staining at 4 weeks after MCT injection also increased. Chronic inhibition of HMGB1 by GLY treatment reduced the MCT-induced increase in right ventricular (RV) systolic pressure, RV hypertrophy (ratio of RV to [left ventricle + septum]), and pulmonary inflammation. MCT-induced muscularization of the pulmonary artery was also attenuated in the GLY-treated group. As assessed 6 weeks after MCT injection, the GLY-treated group exhibited increased survival (90% [18 of 20]) when compared with the control group (60% [12 of 20]; p =0.0027). CONCLUSIONS: Glycyrrhizin, an inhibitor of HMGB1, attenuates pulmonary hypertension progression and pulmonary vascular remodeling in the MCT-induced pulmonary hypertension rat model. Further studies are needed to confirm the potential of HMGB1 as a novel therapeutic target for pulmonary hypertension.

Comparison of endoscopic band ligation and endoclip closure of colonic perforation: technical feasibility and efficacy in an ex vivo pig model.

BACKGROUND AND AIM: Recent reports have indicated several instances of successful treatment of bowel perforation by using endoscopic band ligation (EBL) when treatment with endoclipping is unsuccessful, but this salvage method has not been investigated in any prospective model. Herein we aimed to compare the technical feasibility and efficacy of EBL and endoclip use in intraluminal closure of colon perforation, in an ex vivo model. METHODS: Standardized colonic perforations were created using fresh porcine colon and subsequently closed by full-thickness interrupted sutures, endoclip (QuickClip2(TM)), or EBL. Each closure site was tested with compressed air by using a digital pressure monitor for evaluating leak pressure. RESULTS: No significant differences were noted between the endoclip and EBL in leak pressures. Mean (± SD) pressures for air leakage from the perforations closed using the different devices were as follows: normal colon samples, 52.0 ± 13.2 mmHg; perforations closed with hand-sewn sutures, 32.3 ± 8.3 mmHg; perforations closed with endoclipping, 53.5 ± 22.7 mmHg; and perforations closed with EBL, 50.4 ± 12.5 mmHg. Time taken for closure by EBL was significantly less than that for closure by endoclipping (3.2 ± 1.7 min vs 6.8 ± 1.3 min, P < 0.01). Further, the number of devices used to achieve complete closure in the EBL group was lower than that with endoclipping (1.6 ± 0.5 vs 3.7 ± 0.8, P < 0.01). CONCLUSION: Endoluminal closure of a 1.5-cm colon perforation with EBL decreased procedure time and was not inferior in leak pressure compared with endoclipping in this ex vivo porcine model.

H2 Receptor-Mediated Relaxation of Circular Smooth Muscle in Human Gastric Corpus: the Role of Nitric Oxide (NO).

This study was designed to examine the effects of histamine on gastric motility and its specific receptor in the circular smooth muscle of the human gastric corpus. Histamine mainly produced tonic relaxation in a concentration-dependent and reversible manner, although histamine enhanced contractility in a minor portion of tissues tested. Histamine-induced tonic relaxation was nerve-insensitive because pretreatment with nerve blockers cocktail (NBC) did not inhibit relaxation. Additionally, K(+) channel blockers, such as tetraethylammonium (TEA), apamin (APA), and glibenclamide (Glib), had no effect. However, N(G)-nitro-L-arginine methyl ester (L-NAME) and 1H-(1,2,4)oxadiazolo (4,3-A) quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase (sGC), did inhibit histamine-induced tonic relaxation. In particular, histamine-induced tonic relaxation was converted to tonic contraction by pretreatment with L-NAME. Ranitidine, the H2 receptor blocker, inhibited histamine-induced tonic relaxation. These findings suggest that histamine produced relaxation in circular smooth muscle of human gastric smooth muscle through H2 receptor and NO/sGC pathways.

ASOptimizer: Optimizing antisense oligonucleotides through deep learning for IDO1 gene regulation.

Recent studies have highlighted the effectiveness of using antisense oligonucleotides (ASOs) for cellular RNA regulation, including targets that are considered undruggable; however, manually designing optimal ASO sequences can be labor intensive and time consuming, which potentially limits their broader application. To address this challenge, we introduce a platform, the ASOptimizer, a deep-learning-based framework that efficiently designs ASOs at a low cost. This platform not only selects the most efficient mRNA target sites but also optimizes the chemical modifications for enhanced performance. Indoleamine 2,3-dioxygenase 1 (IDO1) promotes cancer survival by depleting tryptophan and producing kynurenine, leading to immunosuppression through the aryl-hydrocarbon receptor (Ahr) pathway within the tumor microenvironment. We used ASOptimizer to identify ASOs that target IDO1 mRNA as potential cancer therapeutics. Our methodology consists of two stages: sequence engineering and chemical engineering. During the sequence-engineering stage, we optimized and predicted ASO sequences that could target IDO1 mRNA efficiently. In the chemical-engineering stage, we further refined these ASOs to enhance their inhibitory activity while reducing their potential cytotoxicity. In conclusion, our research demonstrates the potential of ASOptimizer for identifying ASOs with improved efficacy and safety.

Changes in postoperative recurrence and prognostic risk factors for patients with gastric cancer who underwent curative gastric resection during different time periods.

BACKGROUND: Current rates of survival for gastric cancer patients are much improved compared with those of the past. The purpose of our study was to analyze the survival of gastric cancer patients according to time period and to examine how different prognostic factors are related to changing survival rates. METHODS: We analyzed data from 7,757 patients who underwent curative gastrectomy after diagnoses of gastric cancer at Samsung Medical Center from 1994 to 2006. Clinicopathologic characteristics and prognostic factors were analyzed retrospectively, with patients divided into period I, from 1994 to 2001, and period II, from 2002 to 2006. RESULTS: The 5-year, disease-free survival rate of patients with gastric cancer increased significantly from 76.7 % during period I to 85.9 % during period II (p < 0.001). The prognosis of the patient who underwent surgery during period I was worse than that of the patient in period II. When multivariate analyses were performed for each time period, independent prognostic factors for period I included patient age >60 years, tumor located in the whole stomach, tumor size, stage, vascular invasion, perineural invasion, and adjuvant chemotherapy. For period II, tumor size, vascular, and adjuvant chemotherapy were no longer independent prognostic risk factors. CONCLUSIONS: The disease-free survival of gastric cancer improved and prognostic factors changed over time. Active, concurrent chemoradiotherapy together with radical gastric resection performed by an expert surgeon seemed to contribute to the improvement in the survival rates of gastric cancer.

A thermophilic alkalophilic α-amylase from Bacillus sp. AAH-31 shows a novel domain organization among glycoside hydrolase family 13 enzymes.

α-Amylases (EC 3.2.1.1) hydrolyze internal α-1,4-glucosidic linkages of starch and related glucans. Bacillus sp. AAH-31 produces an alkalophilic thermophilic α-amylase (AmyL) of higher molecular mass, 91 kDa, than typical bacterial α-amylases. In this study, the AmyL gene was cloned to determine its primary structure, and the recombinant enzyme, produced in Escherichia coli, was characterized. AmyL shows no hydrolytic activity towards pullulan, but the central region of AmyL (Gly395-Asp684) was similar to neopullulanase-like α-amylases. In contrast to known neopullulanase-like α-amylases, the N-terminal region (Gln29-Phe102) of AmyL was similar to carbohydrate-binding module family 20 (CBM20), which is involved in the binding of enzymes to starch granules. Recombinant AmyL showed more than 95% of its maximum activity in a pH range of 8.2-10.5, and was stable below 65 °C and from pH 6.4 to 11.9. The kcat values for soluble starch, γ-cyclodextrin, and maltotriose were 103 s(-1), 67.6 s(-1), and 5.33 s(-1), respectively, and the Km values were 0.100 mg/mL, 0.348 mM, and 2.06 mM, respectively. Recombinant AmyL did not bind to starch granules. But the substitution of Trp45 and Trp84, conserved in site 1 of CBM20, with Ala reduced affinity to soluble starch, while the mutations did not affect affinity for oligosaccharides. Substitution of Trp61, conserved in site 2 of CBM20, with Ala enhanced hydrolytic activity towards soluble starch, indicating that site 2 of AmyL does not contribute to binding to soluble long-chain substrates.

A splinkerette PCR-based genome walking technique for the identification of transgene integration sites in CHO cells.

Identification of recombinant gene integrations sites in the Chinese hamster ovary (CHO) cell genome is increasingly important to assure monoclonality. While next-generation sequencing (NGS) is commonly used for the gene integration site analysis, it is a time-consuming and costly technique as it analyzes the entire genome. Hence, simple, easy, and inexpensive methods to analyze transgene insertion sites are necessary. To selectively capture the integration site of transgene in the CHO genome, we applied splinkerette-PCR (spPCR). SpPCR is an adaptor ligation-based method using splinkerette adaptors that have a stable hairpin loop. Restriction enzymes with high frequencies in the CHO genome were chosen using a Python script and used for the in vitro spPCR assay development. After testing on two CHO housekeeping genes with known loci, the spPCR-based genome walking technique was successfully applied to recombinant CHO cells to identify the transgene integration site. Finally, the comparison with NGS methods exhibited that the time and cost required for the analysis can be substantially reduced. Taken together, the established technique would aid the stable cell line development process by providing a rapid and cost-effective method for transgene integration site analysis.