The role of gastrulation brain homeobox 2 (gbx2) in the development of the ventral telencephalon in zebrafish embryos.

During vertebrate brain development, the gastrulation brain homeobox 2 gene (gbx2) is expressed in the forebrain, but its precise roles are still unknown. In this study, we addressed this issue in zebrafish (Danio rerio) first by carefully examining gbx2 expression in the developing forebrain. We showed that gbx2 was expressed in the telencephalon during late somitogenesis, from 18h post-fertilization (hpf) to 24 hpf, and in the thalamic primordium after 26 hpf. In contrast, another gbx gene, gbx1, was expressed in the anterior-most ventral telencephalon after 36 hpf. Thus, the expression patterns of these two gbx genes did not overlap, arguing against their redundant function in the forebrain. Two-color fluorescence in situ hybridization (FISH) showed close relationships between the telencephalic expression of gbx2 and other forebrain-forming genes, suggesting that their interactions contribute to the regionalization of the telencephalon. FISH further revealed that gbx2 is expressed in the ventricular region of the telencephalon. By using transgenic fish in which gbx2 can be induced by heat shock, we found that gbx2 induction at 16 hpf repressed the expression of emx3, dlx2a, and six3b in the ventral telencephalon. Among secreted factor genes, bmp2b and wnt1 were repressed in the vicinity of the gbx2 domain in the telencephalon. The expression of forebrain-forming genes was examined in mutant embryos lacking gbx2, showing emx3 and dlx2a to be upregulated in the subpallium at 24 hpf. Taken together, these findings indicate that gbx2 contributes to the development of the subpallium through its repressive activities against other telencephalon-forming genes. We further showed that inhibiting FGF signaling and activating Wnt signaling repressed gbx2 and affected the regionalization of the telencephalon, supporting a functional link between gbx2, intracellular signaling, and telencephalon development.

Comprehensive analysis of target genes in zebrafish embryos reveals gbx2 involvement in neurogenesis.

It is well established that the gbx2 homeobox gene contributes to the positioning of the midbrain-hindbrain boundary (MHB) governing the development of adjacent brain regions in vertebrate embryos, but the specific aspects of the gene regulatory network regulated by gbx2 during brain development remain unclear. In the present study, we sought to comprehensively identify gbx2 target genes in zebrafish embryos by microarray analysis around the end of gastrulation, when the MHB is established, using transgenic embryos harboring heat-inducible gbx2. This analysis revealed that a large number of genes were either upregulated or downregulated following gbx2 induction, and the time course of induction differed depending on the genes. The differences in response to gbx2 were found by functional annotation analysis to be related to the functions and structures of the target genes. Among the significantly downregulated genes was her5, whose expression in the midbrain was precisely complementary to gbx2 expression around the MHB, suggesting that gbx2 expression in the anterior hindbrain restricts her5 expression to the midbrain. Because her5 represses neurogenesis, gbx2 may positively regulate neural development in its expression domain. Indeed, we showed further that gbx2 induction upregulated neural marker expression in the midbrain. Quantitative PCR analysis revealed that gbx2 upregulated the expression of the zebrafish proneural gene ebf2, whereas it repressed notch1a, which generally represses neurogenesis. Taken together, these results demonstrate that gbx2 not only functions to position the MHB but also regulates neurogenesis in the anterior hindbrain.

The effectiveness of rapid sequence intubation (RSI) versus non-RSI in emergency department: an analysis of multicenter prospective observational study.

BACKGROUND: Although rapid sequence intubation (RSI) is the method of choice in emergency department (ED) airway management, data to support the use of RSI remain scarce. We sought to compare the effectiveness of airway management between RSI and non-RSI (intubation with sedative agents only or without medications) in the ED. METHODS: Secondary analysis of the data from a multicenter prospective observational registry at 13 Japanese EDs. All non-cardiac-arrest patients who underwent intubation with RSI or non-RSI were included for the analysis. Outcomes of interest were the success rate of intubation and intubation-related complications. RESULTS: Of 2365 eligible patients, 761 (32%) underwent intubations with RSI and 1,604 (68%) with non-RSI. Intubations with RSI had a higher success rate on the first attempt compared to those with non-RSI (73 vs. 63%; P < 0.0001). By contrast, the complication rates did not differ significantly between RSI and non-RSI groups (12 vs. 13%; P = 0.59). After adjusting for age, sex, estimated weight, principal indication, device, specialties and training level of the intubator, and clustering of patients within EDs, intubation with RSI was associated with a significantly higher success rate on the first attempt (OR, 2.3; 95% CI, 1.8-2.9; P < 0.0001) while that with RSI was not associated with the risk of complications (OR, 0.9; 95% CI, 0.6-1.2; P = 0.31). CONCLUSIONS: In this large multicenter study of ED airway management, we found that intubation with RSI was independently associated with a higher success rate on the first attempt but not with the risk of complications.

Caring for patients with malignant pleural mesothelioma in Japan: evaluation of a Palliative Care Educational Program.

PURPOSE: This study evaluated the effect of an Educational PROGRAM on Palliative Care for MPM for Nurses in Japan. PROGRAM: The 5-h program consisted of lectures and care planning group work. MATERIALS AND METHODS: This study used a pretest-posttest design with a single cohort of nurses and included a Difficulties in Palliative Care for Patients with MPM (DPCMPM) Scale with 15 items. The pre- and posttest scores were compared using a t-test. RESULTS: We included 27 female nurses with a mean of 14.4 years of nursing experience. In 12 of 15 DPCMPM items, the posttest difficulty scores were lower than the pretest scores. Participants highly evaluated the program for validity, clarity, clinical usefulness, and the facilitators. The Palliative Care for MPM Handbook for Nurses was developed as an educational tool for clinical settings. CONCLUSIONS: The Educational PROGRAM on Palliative Care for MPM for Nurses was effective in reducing nursing difficulties.

Gbx2 functions as a transcriptional repressor to regulate the specification and morphogenesis of the mid-hindbrain junction in a dosage- and stage-dependent manner.

The Gbx subfamily of homeodomain transcription factors is involved in the positioning of the isthmus, which patterns the midbrain and cerebellum in vertebrates. To uncover the details of Gbx functions, we first examined the dose dependency of its effects on brain formation in zebrafish and found that high-dose gbx2 mRNA injection affected the entire forebrain and midbrain, whereas low-dose mRNA specifically disrupted the isthmic folding at the midbrain-hindbrain boundary (MHB) but only weakly affected the expression of genes involved in MHB specification. Thus, isthmus morphogenesis, and not its early specification, is highly sensitive to gbx2. Transient induction of heat-inducible gbx2 using transgenic fish showed that MHB specification is most sensitive to gbx2 at the end of epiboly and further suggested that otx2 is the direct target gene. These together demonstrate that gbx2 regulates both specification and morphogenesis of the MHB/isthmus region. Deletion analyses showed that both the N- and C-terminal regions contribute to the suppressive activity of Gbx2 against the anterior brain and that the N-terminal core region, including the Eh1 and proline-rich sequences, is required for this Gbx2 activity. Comparison of the effects of activated and repressive forms with wild-type Gbx2 suggested that Gbx2 functions as a transcriptional repressor, which was further evidenced by a luciferase assay in which gbx2 repressed the MHB enhancer of fgf8a in mouse P19 cells.

[Application of a rapid and simple multi-residue method for determination of pesticide residues in drinking water and beverages using liquid chromatography-tandem mass spectrometry].

A rapid and simple multi-residue method for determination of pesticides has been applied to drinking water and beverages. To a disposable polypropylene tube containing 10.0 g sample, 20 mL acetonitrile was added and the mixture was shaken vigorously for 1 min to extract pesticides. Then, 1 g sodium chloride and 4 g magnesium sulfate anhydrous were added, followed by vigorous shaking for 1 min and centrifugation to obtain the organic phase. The organic phase was processed with a graphite carbon black/PSA solid phase column. After concentration and reconstitution with 25% methanol containing aqueous solution, the test solution was analyzed with LC-MS/MS. Recovery tests of 91 pesticides fortified (0.02 µg/g) in 35 kinds of drinking water and beverages were conducted. The decline of recoveries in alcoholic beverages is considered to be due to the increase of organic phase volume owing to ethanol included in the alcoholic beverages. A simulation study was carried out with simulated alcoholic beverages, which consisted of 50% grape juice, with various amounts of ethanol and water, to examine pesticides recoveries and volume of the organic phase. The results suggested this method would be applicable both to alcoholic beverages containing less than 10% ethanol and to alcoholic beverages containing over 10% ethanol after dilution with water to below 10% ethanol prior to the addition of acetonitrile. A sample could be processed and analyzed by LC-MS/MS within 2 h. Thus, this method should be useful for monitoring and screening pesticide residues in drinking water and various beverages.

Pou2, a class V POU-type transcription factor in zebrafish, regulates dorsoventral patterning and convergent extension movement at different blastula stages.

Zebrafish pou2, which encodes a class V POU transcription factor considered to be an orthologue of mouse Oct-3/4, has been implicated by mutant analysis in dorsoventral (DV) patterning, gastrulation, and endoderm formation in early embryos and later in the regionalization of the neural plate. A series of gain-of-function experiments were conducted in the present study to directly reveal the roles pou2 plays in embryogenesis. We first revealed that injecting low-dose wild-type pou2 mRNA ventralizes embryos. Similar overexpression of activated (vp-) pou2 resulted in the same effects, whereas repressive (en-) pou2 caused dorsalization, supporting the previously proposed idea that pou2 is involved in DV patterning and that pou2 is a transcriptional activator. In contrast, high-dose mRNA for pou2 and its modified genes affected convergent extension (CE) movement. We observed similar activities for mouse Oct-3/4, suggesting conservation of the roles of this POU family in vertebrate development. To determine the critical stage for the functions of pou2 in embryos, we established a transgenic (Tg) fish line harboring en-pou2 under regulation of a heat-shock promoter (HEP) and found that the exposure of HEP Tg embryos to heat shock at the midblastula (sphere) stage dorsalized embryos, whereas induction of HEP at the late blastula stage (30-50% epiboly) affected CE movement. The defects due to HEP induction were rescued by introducing wild-type pou2 mRNA before the heat treatments. Collectively, these data demonstrated that pou2 regulates DV patterning and CE movement in zebrafish embryos at the midblastula and late blastula stages, respectively.

Mesendoderm specification depends on the function of Pou2, the class V POU-type transcription factor, during zebrafish embryogenesis.

Zebrafish pou2, encoding the class V POU transcription factor orthologous to mouse Oct-3/4, has been implicated in different aspects of development, such as dorsoventral patterning, endoderm formation, and brain regionalization, by analyzing pou2 mutant embryos. In the present study, we first conducted overexpression of pou2-modified genes by mRNA injection, and found that pou2 and its active form (vp-pou2) augmented mesoderm formation and suppressed endoderm specification, whereas repressive pou2 (en-pou2) affected the formation of the mesoderm and endoderm in a different manner. To avoid complications that might arise from different pou2 functions during the course of development, we used a transgenic line harboring inducible en-pou2 (HEP), which could function in a dominant-negative manner. We found that suppressing endogenous pou2 by HEP induction at the mid-blastula stage enhanced endoderm development at the expense of mesoderm, whereas the same treatment in the late blastulae promoted mesoderm formation and suppressed the endoderm specification. Further analyses using HEP induction revealed that, from late epiboly to early somitogenesis, pou2 regulated additional developmental aspects, such as brain regionalization, heart development, and tail formation. Our findings suggest that Pou2 functions in multiple aspects of vertebrate development, especially in the binary decision of the mesendoderm to mesoderm and endoderm in different ways depending on the developmental stage.

[Preparation of samples for proficiency testing of pesticide residue analysis in processed foods].

To conduct proficiency testing for the analysis of pesticide residues in processed foods, fortified samples of retort curry and pancake were examined. In the case of retort curry, heating and mixing were necessary at the time of preparation to provide a homogenous analytical sample. A mixture of 4 carbamates and 11 organophosphorus pesticides was spiked and 14 of them showed consistent results in the samples. In the case of pancake, 10 kinds of pesticides were added to the pastry. The prepared pastry was them cooked. The relative concentrations of most of the pesticides in the pancake were not affected and all the pesticides showed consistent results in the samples. These results showed that the two tested samples were suitable for proficiency testing.

FGF receptor gene expression and its regulation by FGF signaling during early zebrafish development.

The expression of all four fgfr genes was extensively examined throughout early embryogenesis of the zebrafish (Danio rerio). fgfr1 alone was expressed maternally throughout the blastoderm, and then zygotically in the anterior neural plate and presomitic mesoderm. fgfr4 expression was first detected in late blastulae and was gradually restricted to the brain. fgfr2 and fgfr3 expression were initiated in early and late gastrulae, respectively; fgfr2 was expressed in the anterior neural plate and somitic mesoderm, whereas fgfr3 was activated in the axial mesoderm and then in the midbrain and somitic mesoderm. During somitogenesis, each of these fgfr genes was expressed in a characteristic manner in the brain. Using an FGF signal inhibitor, dominant-negative FGF receptors and fgf8.1/fgf8a mutants, we found that fgfr expression is directly or indirectly regulated by FGF signaling during epiboly and at the end of somitogenesis, revealing the presence of an autoregulatory mechanism.

Actual and estimated costs of disposable materials used during surgical procedures.

It is difficult to estimate precisely the costs of disposable materials used during surgical operations. To evaluate the actual costs of disposable materials, we calculated the actual costs of disposable materials used in 59 operations by taking account of costs of all disposable materials used for each operation. The costs of the disposable materials varied significantly from operation to operation (US$ 38-4230 per operation), and the median [25-percentile and 75-percentile] of the sum total of disposable material costs of a single operation was found to be US$ 686 [205 and 993]. Multiple regression analysis with a stepwise regression method showed that costs of disposable materials significantly correlated only with operation time (p<0.001). Based on the results, we propose a simple method for estimating costs of disposable materials by measuring operation time, and we found that the method gives reliable results. Since costs of disposable materials used during surgical operations are considerable, precise estimation of the costs is essential for hospital cost accounting. Our method should be useful for planning hospital administration strategies.

Relief of oxidative stress by ascorbic acid delays cellular senescence of normal human and Werner syndrome fibroblast cells.

Primary human cells have a definite life span and enter into cellular senescence before ceasing cell growth. Oxidative stress produced by aerobic metabolism has been shown to accelerate cellular senescence. Here, we demonstrated that ascorbic acid, used as an antioxygenic reagent, delayed cellular senescence in a continuous culture of normal human embryonic cells, human adult skin fibroblast cells, and Werner syndrome (WS) cells. The results using human embryonic cells showed that treatment with ascorbic acid phospholic ester magnesium salt (APM) decreased the level of oxidative stress, and extended the replicative life span. The effect of APM to extend the replicative life span was also shown in normal human adult cells and WS cells. To understand the mechanism of extension of cellular life span, we determined the telomere lengths of human embryonic cells, both with and without APM treatment, and demonstrated that APM treatment reduced the rate of telomere shortening. The present results indicate that constitutive oxidative stress plays a role in determining the replicative life span and that suppression of oxidative stress by an antioxidative agent, APM, extends the replicative life span by reducing the rate of telomere shortening.

Impact of antithrombin deficiency in thrombogenesis: lipopolysaccharide and stress-induced thrombus formation in heterozygous antithrombin-deficient mice.

Antithrombin (AT) deficiency is an autosomal disorder associated with venous thromboembolism. However, a diagnosis of homozygous AT deficiency is seldom made. Most patients are heterozygous and have approximately 50% AT activities, and they are at higher risk for the development of thromboembolism. Through gene targeting we generated AT-deficient mice and previously reported that completely AT-deficient mice could not survive the prenatal period because of extensive thrombosis in the myocardium and liver sinusoids. In contrast, heterozygous AT-deficient mice with 50% AT activities have not shown spontaneous thromboembolic episodes. To demonstrate a thrombotic tendency in heterozygous AT deficiency, we challenged heterozygous AT-deficient mice (AT+/- mice) with the administration of lipopolysaccharide (LPS) or with restraint stress by immobilization. LPS injection markedly induced fibrin deposition in the kidney glomeruli, myocardium, and liver sinusoids in AT+/- mice compared with wild-type mice (AT+/+ mice). Restraint stress tests were performed by placing mice in 50-mL conical centrifuge tubes for 20 hours. Fibrin deposition was observed in the kidney of AT+/+ and AT+/- mice, but AT+/- mice exhibited more extensive fibrin deposition than AT+/+ mice. After prophylactic administration of human AT concentrates to increase plasma AT activities of AT+/- mice, LPS-induced fibrin deposition was effectively prevented. These results suggest that heterozygous AT deficiency is significantly associated with a tendency toward thrombosis formation in the kidney. The AT+/- mouse thus is a useful model for studying the effect of environmental or genetic risk factors on thrombogenesis.