A web-based self-learning system for ultrasound-guided vascular access.

Ultrasound-guided vascular access is practiced widely. Optimal educational methods have not yet been established. We hypothesized that a step-by-step web-based learning system is effective for self-learning. In this study, we examined the potential of this system as a self-learning tool. This was an observational study at a single institution. Participants included residents, who were self-educated through the web-based system. Skill proficiency was measured after self-learning. The primary outcome was the extent to which self-learning enabled residents to acquire proficiency in the basic skills of ultrasound-guided vascular access: needle visualization, hand-eye coordination, and avoiding posterior wall penetration. A secondary outcome was the time required to achieve proficiency. Thirty-nine residents were enrolled in this study. Eleven residents (28%) passed the first skill assessment test. There was no significant difference in the number of days that the web-based system was accessed, the total number of screen views, or the total learning time between participants who passed and those who failed the first test. Skill assessment scores between those who passed and those who failed the first test were different, especially the score for hand-eye coordination, and the number of posterior wall penetrations. Self-learning with a web-based system enabled 28% of residents to pass the first skill assessment test. The remaining 72% failed the first skill assessment test but continued to learn using the web-based system and eventually passed the test. Hence, the web-based system needed formative testing to function as a self-learning system. Simulation education for vascular access is expected to increase in educational content and methods. Self-learning through a web-based learning system is a leading candidate for this growth.

Post-surgical pseudoaneurysm of the corona mortis artery treated by arterial embolization from two arteries: A case report.

Post-surgical pseudoaneurysm in the pelvis is rare. However, when it does occur, it may cause life-threatening hemorrhage. Hemostatic treatment for pelvic pseudoaneurysms may be complicated because the blood vessels in the pelvis may present with various anastomoses. Herein, we describe a case of a pseudoaneurysm that necessitated embolization of two arteries. A 47-year-old woman had undergone a total hysterectomy, a bilateral adnexectomy, and a pelvic lymphadenectomy for endometrial cancer; 13 days after surgery, she complained of sudden abdominal pain. Contrast-enhanced computed tomography revealed a retroperitoneal hematoma and a pseudoaneurysm with contrast leakage. The pseudoaneurysm had two feeding arteries (from the external and internal iliac systems). The first feeding artery was the obturator artery, which arose from the anterior trunk of the internal iliac artery. The second feeding artery was the aberrant obturator artery, which arose from the medial femoral circumflex artery. Both feeders were embolized and hemostasis was achieved. Pseudoaneurysms in the pelvis may have double origins from the external and internal iliac systems, and the aberrant obturator artery may arise from the medial femoral circumflex artery. Therefore, radiologists should be aware of these variations to effectively address post-surgical pseudoaneurysms of the corona mortis artery.

Mutation in Sodium-Glucose Cotransporter 2 Results in Down-Regulation of Amyloid Beta (A4) Precursor-Like Protein 1 in Young Age, Which May Lead to Poor Memory Retention in Old Age.

Senescence-accelerated mouse prone 10 (SAMP10) exhibits cerebral atrophy and depression-like behavior. A line of SAMP10 with spontaneous mutation in the Slc5a2 gene encoding the sodium-glucose cotransporter (SGLT) 2 was named SAMP10/TaSlc-Slc5a2slc (SAMP10-ΔSglt2) and was identified as a renal diabetes model. In contrast, a line of SAMP10 with no mutation in SGLT2 (SAMP10/TaIdrSlc, SAMP10(+)) was recently established under a specific pathogen-free condition. Here, we examined the mutation effect in SGLT2 on brain function and longevity. No differences were found in the survival curve, depression-like behavior, and age-related brain atrophy between SAMP10-ΔSglt2 and SAMP10(+). However, memory retention was lower in SAMP10-ΔSglt2 mice than SAMP10(+). Amyloid beta (A4) precursor-like protein 1 (Aplp1) expression was significantly lower in the hippocampus of SAMP10-ΔSGLT2 than in SAMP10(+) at 2 months of age, but was similar at 12 months of age. CaM kinase-like vesicle association (Camkv) expression was remarkably lower in SAMP10(+). These genes have been reported to be involved in dendrite function. Amyloid precursor proteins have been reported to involve in maintaining homeostasis of glucose and insulin. These results suggest that mutation in SGLT2 results in down-regulation of Aplp1 in young age, which can lead to poor memory retention in old age.

Serum Magnesium Levels and Neurological Outcomes in Patients Undergoing Targeted Temperature Management After Cardiac Arrest.

INTRODUCTION: Magnesium plays a neuroprotective role at the physiologic level, but its neuroprotective role in patients undergoing targeted temperature management for cardiac arrest is not well established. We performed multiple logistic regression analysis to evaluate whether magnesium levels can predict neurological outcomes in patients undergoing targeted temperature management after cardiac arrest. METHODS: We retrospectively investigated data on 86 patients who had undergone targeted temperature management after cardiac arrest between December 2015 and November 2017. The primary outcome was to determine whether magnesium levels predict unfavorable neurological outcomes for patients with return of spontaneous circulation after targeted temperature management. Cerebral Performance Category 3, 4, or 5 indicated unfavorable neurological outcomes. We performed multiple logistic regression to evaluate the primary outcome, adjusting for the time to return of spontaneous circulation, motor score of the Glasgow Coma Scale, first-recorded cardiac rhythm, pH, and magnesium levels. RESULTS: Of the 86 patients, 58 had unfavorable neurological outcomes. The mean hospital stay was 19 days. Multivariable analysis indicated that magnesium levels were not associated with an unfavorable neurological outcome. In contrast, a time to return of spontaneous circulation greater than 30 minutes and Glasgow Coma Scale motor score of 1 were significantly associated with an unfavorable neurological outcome. DISCUSSION: Magnesium levels were not associated with an unfavorable neurological outcome according to multivariable analysis. We found that a time to return of spontaneous circulation greater than 30 minutes and Glasgow Coma Scale motor score of 1 might predict an unfavorable neurological outcome.

Stomal stenosis during gradual closure of a traumatic abdominal wall hernia.

INTRODUCTION: Traumatic abdominal wall hernias are often accompanied by intra-abdominal injuries, and a stoma may be required. Although rare, stomal stenosis can develop after the repair of a traumatic abdominal wall hernia. PRESENTATION OF CASE: A 65-year-old woman was in a head-on collision with a truck and was brought by ambulance to our facility. The findings of a physical examination and computed tomography scan suggested bowel perforation for which exploratory surgery was performed. The lacerated small bowel and sigmoid colon were resected and an ileostomy and colostomy were created. Abdominal wall reconstruction was impossible because of the large defect size. Repair of the abdominal wall was achieved by gradual closure of the fascia after surgery in combination with negative pressure wound therapy. Stenosis of the ileostomy occurred during this process and was surgically repaired. DISCUSSION: We reconstructed the abdominal wall using negative pressure wound therapy in combination with sutures while minimizing the risk of abdominal compartment syndrome. This approach did not increase the intra-abdominal pressure, but it deformed the abdominal wall, resulting in unexpected stenosis of the ostomy. CONCLUSION: Gradual postoperative closure of a traumatic abdominal wall hernia with an ostomy in place may result in stomal stenosis. Stomal patency must be carefully evaluated during this process.

Essential Role of NADPH Oxidase-Dependent Production of Reactive Oxygen Species in Maintenance of Sustained B Cell Receptor Signaling and B Cell Proliferation.

Reactive oxygen species (ROS) are not only toxic substances inducing oxidative stress but also play a role as a second messenger in signal transduction through various receptors. Previously, B cell activation was shown to involve prolonged ROS production induced by ligation of BCR. However, the mechanisms for ROS production and ROS-mediated activation in B cells are still poorly understood. In this study, we demonstrate that BCR ligation induces biphasic ROS production in both mouse spleen B cells and the mouse B cell line BAL17; transient and modest ROS production is followed by sustained and robust ROS production at 2-6 h after BCR ligation. ROS production in the late phase but not in the early phase augments activation of signaling pathways, such as the NF-κB and PI3K pathways, and is essential for B cell proliferation. ROS production in the late phase appears to be mediated by NADPH oxidases (NOXes) because prolonged ROS production is inhibited by various NOX inhibitors, including the specific inhibitor VAS2870. BCR ligation-induced ROS production is also inhibited by CRISPR/Cas9-mediated deletion of either the Cyba gene encoding p22phox, the regulator of NOX1-4 required for their activation, or NOX3, whereas ROS production is not affected by double deficiency of the DUOXA1 and DUOXA2 genes essential for the activation of the NOX isoforms DUOX1 and DUOX2. These results indicate that NOXes play a crucial role in sustained but not early BCR signaling and suggest an essential role of NOX-dependent sustained BCR signaling in B cell activation.

Hypoxia induces CD133 expression in human lung cancer cells by up-regulation of OCT3/4 and SOX2.

CD133 has been recognized as a specific cell surface marker for cancer stem cells in various tumors, although its biological functions and transcriptional regulation remain unclear. We found that the CD133 expression level was up-regulated in the lung cancer cell lines N417, H358, and A549, when these cell lines were cultured under hypoxic conditions. Among the five promoters (P1-P5) of human CD133 gene loci, P1 promoter was most strongly associated with hypoxia-induced promoter activity of CD133 gene expression. The P1 promoter possesses several cis-regulatory elements, including RUNT, GATA, ETS, OCT, SRY, and CREB-binding sites. A series of deletion and base substitution mutants of the P1 promoter revealed that OCT- and SRY-binding sites are important for hypoxia-induced promoter activity. The chromatin immunoprecipitation assay further confirmed the direct binding of Octamer biding trans-cription factor 3/4 (OCT4) and/or SRY-box containing gene 2 (SOX2) to the P1 promoter region of CD133 gene loci. In addition, the enhancement of both OCT4 and SOX2 expression by the α subunit of hypoxia-inducible factors (HIF1α and HIF2α) was required for hypoxia-induced CD133 expression. Knockdown of OCT4 or SOX2 expression in N417 cells with stabilized HIF1α and/or HIF2α abolished CD133P1 activity, while ectopic OCT4 or SOX2 expression triggers CD133P1 activity in the absence of HIF1α or HIF2α. Thus, in the hypoxic conditions, OCT4 and SOX2, both of which are induced by HIF1α/HIF2α. promote CD133 expression in the lung cancer cells via their direct interaction with the P1 promoter.

Physical and functional interactions between hematopoietic cell-specific ETS transcription factors and homeodomain proteins.

To examine the possibility that ETS family transcription factors, PU.1, SPI-B, ELF-1, ERG-3, ETS-1 and TEL, and homeodomain proteins, HOXA10, HOXC13, MEIS1 and PBX1B, function cooperatively, we investigated their interactions. In luciferase assays, HOXA10 and HOXC13 augmented the activity of PU.1 and SPI-B while diminishing that of ELF-1 and ERG-3. MEIS1 diminished the activity of ETS-1. No clear effects were observed for other combinations. Immunoprecipitation assays showed protein-protein interactions among the combinations exhibiting functional interactions. A mutation of HOXC13, which abolished binding to ELF-1, also abolished the diminishing effect on ELF-1. The results suggest functional interaction through physical interactions.

The Arabidopsis OBERON1 and OBERON2 genes encode plant homeodomain finger proteins and are required for apical meristem maintenance.

Maintenance of the stem cell population located at the apical meristems is essential for repetitive organ initiation during the development of higher plants. Here, we have characterized the roles of OBERON1 (OBE1) and its paralog OBERON2 (OBE2), which encode plant homeodomain finger proteins, in the maintenance and/or establishment of the meristems in Arabidopsis. Although the obe1 and obe2 single mutants were indistinguishable from wild-type plants, the obe1 obe2 double mutant displayed premature termination of the shoot meristem, suggesting that OBE1 and OBE2 function redundantly. Further analyses revealed that OBE1 and OBE2 allow the plant cells to acquire meristematic activity via the WUSCHEL-CLAVATA pathway, which is required for the maintenance of the stem cell population, and they function parallel to the SHOOT MERISTEMLESS gene, which is required for preventing cell differentiation in the shoot meristem. In addition, obe1 obe2 mutants failed to establish the root apical meristem, lacking both the initial cells and the quiescent center. In situ hybridization revealed that expression of PLETHORA and SCARECROW, which are required for stem cell specification and maintenance in the root meristem, was lost from obe1 obe2 mutant embryos. Taken together, these data suggest that the OBE1 and OBE2 genes are functionally redundant and crucial for the maintenance and/or establishment of both the shoot and root meristems.

Two of four alternatively spliced isoforms of RUNX2 control osteocalcin gene expression in human osteoblast cells.

Runx2 is a Runt domain transcription factor that transcriptionally regulates osteoblast differentiation and bone formation. In this study, we show that human chondro- and osteosarcoma cell lines, human mesenchymal stem cells (hMSC) and a human primary chondrocytes (HC), osteoblst cells (HOb) express an intact isoform (RUNX2wt) and 3 alternatively spliced isoforms (RUNX2Delta5, Delta7, and Delta5Delta7) that are generated by skipping exon 5 and/or exon 7. Two of the truncated forms of RUNX2 (RUNX2Delta5 and RUNX2Delta5Delta7) did not localize in the nucleus and had lost their DNA binding activity. In cotransfection experiments with an osteocalcin (OC) promoter construct, we confirmed that only RUNX2wt and RUNX2Delta7 could upregulate the OC promoter activity in the osteosarcoma cell line. In addition, the coactivator CBP/p300 enhanced the transcriptional activity of the OC promoter when coexpressed with RUNX2wt or RUNX2Delta7, but not when coexpressed with RUNX2Delta5 or RUNX2Delta5Delta7. In contrast, the corepressor HDAC3 only repressed the activation from the OC promoter when coexpressed with RUNX2wt. These results support the hypothesis that RUNX2 both up- and downregulates its target gene promoters, as exemplified by the OC gene, using various isoforms and context-dependent formation of transcriptional complexes.

Interaction between the homeodomain protein HOXC13 and ETS family transcription factor PU.1 and its implication in the differentiation of murine erythroleukemia cells.

Some of homeodomain proteins and the ETS family of transcription factors are involved in hematopoiesis. RT-PCR analysis revealed that the HOXC13 and PU.1 genes were expressed in murine erythroleukemia (MEL) cells and their levels decreased during DMSO-induced differentiation into erythroid cells. HOXC13 bound to the ETS domain of PU.1 through a region encompassing the C-terminal part of the homeodomain and the most C-terminal region and enhanced the transcriptional activity of PU.1. Enforced expression of HOXC13 in MEL cells resulted in the suppression of beta-globin gene expression. In MEL cells overexpressing HOXC13 and PU.1, which also inhibits the differentiation of MEL cells, no synergistic effect on the suppression of beta-globin gene expression was observed. However, in the presence of DMSO, the expression levels of the beta-globin gene in the cells overexpressing HOXC13 and PU.1 were, unexpectedly, higher than those in the cells overexpressing PU.1 alone. The levels of PU.1 protein were markedly decreased despite that the levels of mRNA were preserved in the cells overexpressing PU.1 and HOXC13. It was, thus, suggested that although HOXC13 negatively regulates the differentiation of MEL cells into erythroid cells, it antagonizes PU.1 possibly by down-regulation of PU.1 protein in the presence of a differentiation stimulus.

CRM1/BIG-mediated auxin action regulates Arabidopsis inflorescence development.

The shape of the inflorescence in Arabidopsis thaliana ecotype Columbia is a raceme with individual flowers developing acropetally. The ecotype Landsberg harboring the erecta (er) mutation shows a corymb-like inflorescence, namely a compact inflorescence with a flattened arrangement of flower buds at the tip. To gain insight into inflorescence development, we previously isolated corymb-like inflorescence mutants, named corymbosa1 (crm1), and found that the corymb-like inflorescence in crm1-1 was due to reduced cell elongation of pedicels and stem internodes. Double mutants of crm1 with er and crm2, and crm1-1 crm2-1 er-105 triple mutants show an additive phenotype. crm1-1 is caused by a mutation in BIG, which is required for polar auxin transport. CRM1/BIG is expressed in inflorescence meristems, floral meristems and vascular tissues. We analyzed a collection of 12 reduced lateral root formation (rlr) mutants, which are allelic to crm1-1, and categorized the mutants into three classes, depending on the plant developmental defects. Although all 12 alleles had new stop codons, the phenotype of heterozygous crm1-1/doc1-1 and Northern blotting suggest that new crm1/big mutant alleles are hypomorphic. Auxin-responsive DR5rev::GFP expression was decreased in crm1-1 vasculature of pedicels and stem internodes. PINFORMED1 (PIN1) and CRM1/BIG are expressed in vasculature of pedicels and stem internodes. The severity of corymb-like inflorescence in crm1/big mutants correlated with increased levels of PIN1. Our results suggest that CRM1/BIG controls the elongation of the pedicels and stem internodes through auxin action.

Impaired repressor activity and biological functions of PU.1 in MEL cells induced by mutations in the acetylation motifs within the ETS domain.

PU.1, a hematopoietic Ets transcription factor, is required for development of the lymphoid and myeloid lineages. We have previously shown that PU.1 functions as both a transcriptional activator and repressor through complex formation with CBP/p300 and HDAC1/mSin3A/MeCP2, respectively. To determine whether modification of PU.1 is responsible for switching its association between co-activators and co-repressors, we examined whether acetylation regulates the physical and functional activities of PU.1. PU.1 was acetylated in vivo and its repressor activity was reduced when the putative acetylation motifs in the Ets domain were mutated. The mutant cooperated with CBP similar to wild type PU.1, but insufficiently with GATA-1 and mSin3A. Whereas overexpression of wild type PU.1 induced differentiation block, growth inhibition, and apoptotic cell death in MEL erythroleukemia cells as we reported previously, overexpression of the mutant-acetylation motif PU.1 did not. Taken together, our data suggest that acetylation might regulate the biological functions of PU.1 in erythroid cells.

Effects of PU.1-induced mouse calcium-calmodulin-dependent kinase I-like kinase (CKLiK) on apoptosis of murine erythroleukemia cells.

PU.1, a hematopoietic cell-specific Ets family transcription factor, is involved in the generation of murine erythroleukemia (MEL). To identify the target gene(s) of PU.1 in MEL cells, we carried out differential display (DD) analysis and isolated a novel gene whose expression was up-regulated after overexpression of PU.1 in MEL cells. Because the gene exhibited about 90% homology with the human calcium-calmodulin-dependent kinase I-like kinase (CKLiK) gene, it was identified as a mouse homologue of human CKLiK. The mCKLiK gene was mapped to the mouse chromosome 2A1-A3 region and shown to be expressed predominantly in T cells lymphoma and embryonal carcinoma cell lines and primary thymus and brain. Two types of transcripts were present showing a difference in the 3' portion of the coding region and CREB-activating ability. Overexpression of each isoform of mCKLiK in MEL cells revealed that one of them induces, while the other inhibits apoptosis under low serum condition. Differentiation inhibition and lineage switch to myelomonocytes, which were previously observed in MEL cells overexpressing PU.1, were not provoked in the cells overexpressing mCKLiK. These results suggest that mCKLiK is up-regulated by PU.1 in MEL cells and involved in apoptosis of the cells.

Direct association between PU.1 and MeCP2 that recruits mSin3A-HDAC complex for PU.1-mediated transcriptional repression.

PU.1, a member of the Ets family of transcription factors, is implicated in hematopoietic cell differentiation through its interactions with other transcriptional factors and cofactors. To identify a novel protein(s) binding to PU.1, we carried out affinity purification using a column of Glutathione-Sepharose beads bound to GST-PU.1 fusion protein and isolated several individual proteins using murine erythroleukemia (MEL) cell extracts. Sequence analysis of these proteins revealed that one was MeCP2 a methyl CpG binding protein. GST-pull-down assay and immunoprecipitation assay showed that PU.1 bound directly to MeCP2 via its Ets domain and MeCP2 bound to PU.1 via either its amino terminal domain or trans-repression domain. MeCP2 repressed transcriptional activity of PU.1 on a reporter construct with trimerized PU.1 binding sites. This downregulation was recovered in the presence of histone deacetylase inhibitor, trichostatin A (TSA). MeCP2 was integrated in PU.1-mSin3A-HDAC complex but not in PU.1-CBP complex. Chromatin immunoprecipitation (ChIP) assays showed that PU.1 and MeCP2 were collocated at the PU.1 binding site on the reporter construct and the PU.1 binding site of the intervening sequence 2 (IVS2) region in the intron of the beta-globin gene, which has been proposed to regulate expression of the gene, in undifferentiated MEL cells. The complex disappeared from the region during the course of erythroid differentiation of MEL cells. Our results suggest that MeCP2 acts as a corepressor of PU.1 probably due to facilitating complex formation with mSin3A and HDACs.

Formation of corymb-like inflorescences due to delay in bolting and flower development in the corymbosa2 mutant of Arabidopsis.

Among the wild-type ecotypes of Arabidopsis thaliana whose shape of inflorescence is categorized as raceme, the ecotype Landsberg harboring the erecta (er) mutation shows a corymb-like inflorescence, namely, a compact inflorescence with a flattened arrangement of flower buds at the tip. The fact that the ER gene encodes a receptor-like protein kinase implies the presence of a signaling cascade responsible for the inflorescence morphology of flowering plants. We report here the characterization of another mutant with a corymb-like inflorescence, named corymbosa2 (crm2), and the isolation of the CRM2 gene. While the er mutation causes a severe reduction in the length of pedicels, the crm2 mutation results in a significant delay in the initiation of internode elongation and in the development of flowers, despite having little effect on the timing of floral induction. Consequently, the number of flower buds is apparently increased at the tip of crm2 inflorescence. The crm2 er double mutant shows an additive phenotype. These results suggest that CRM2 and ER may act in different ways to generate wild-type inflorescence. The CRM2 gene was isolated by positional cloning and appears to encode a polypeptide with no significant homology to known sequences.

Interaction between the hematopoietic Ets transcription factor Spi-B and the coactivator CREB-binding protein associated with negative cross-talk with c-Myb.

We have previously shown that the hematopoietic Ets transcription factor PU.1 interacts with the transcriptional coactivator CREB-binding protein (CBP). In this study, we further investigated whether Spi-B, another hematopoietic Ets transcription factor, also interacts with CBP. Direct physical interaction of Spi-B with CBP was demonstrated by glutathione S-transferase binding assay. Analysis using several deletion mutants of Spi-B and CBP revealed that the NH2-terminal region including the activation domain of Spi-B interacted with the region spanning amino acid residues 1283-1915 of CBP in vitro. The interaction of Spi-B with CBP was also observed in vivo. CBP potentiated Spi-B-mediated transcription of the reporter gene driven by the multimerized PU.1/Spi-B binding sites. This transcriptional activation by Spi-B and CBP was inhibited by expression of c-Myb, and the transcriptional activation by c-Myb and CBP was inhibited by expression of Spi-B, suggesting competition for CBP between these two transcription factors. Our results suggest that CBP acts as a transcriptional coactivator of Spi-B and mediates synergistic or antagonistic interactions between other transcription factors.