Vascularized Bone-Mimetic Hydrogel Constructs by 3D Bioprinting to Promote Osteogenesis and Angiogenesis.

Bone is a highly vascularized tissue with a unique and complex structure. Long bone consists of a peripheral cortical shell containing a network of channels for vascular penetration and an inner highly vascularized bone marrow space. Bioprinting is a powerful tool to enable rapid and precise spatial patterning of cells and biomaterials. Here we developed a two-step digital light processing technique to fabricate a bone-mimetic 3D hydrogel construct based on octacalcium phosphate (OCP), spheroids of human umbilical vein endothelial cells (HUVEC), and gelatin methacrylate (GelMA) hydrogels. The bone-mimetic 3D hydrogel construct was designed to consist of a peripheral OCP-containing GelMA ring to mimic the cortical shell, and a central GelMA ring containing HUVEC spheroids to mimic the bone marrow space. We further demonstrate that OCP, which is evenly embedded in the GelMA, stimulates the osteoblastic differentiation of mesenchymal stem cells. We refined the design of a spheroid culture device to facilitate the rapid formation of a large number of HUVEC spheroids, which were embedded into different concentrations of GelMA hydrogels. It is shown that the concentration of GelMA modulates the extent of formation of the capillary-like structures originating from the HUVEC spheroids. This cell-loaded hydrogel-based bone construct with a biomimetic dual ring structure can be potentially used for bone tissue engineering.

[INVESTIGATION OF SUBCUTANEOUS IMMUNOTHERAPY FOR ALLERGIC RHINITIS IN OUR HOSPITAL FOR THE PAST 17 YEARS].

BACKGROUND: Recently, subcutaneous immunotherapy (SCIT) has not been performed very much in our country. We investigated SCIT for allergic rhinitis in our hospital for the past 17 years. METHODS: Subjects were 188 cases who started SCIT by rush method under hospitalization. We investigated the patient background, laboratory findings, side reactions from their medical records and carried out questionnaire survey about effects and satisfaction to patients and their guardians. RESULTS: The median age at start was 10 years 4 months. Cedar pollen was the most common allergen used in treatment followed by mite, and two or more allergens were used in 85% patients. Five percent of patients dropped out. Medication score decreased significantly after SCIT, and 45% had become free from medication. Systemic adverse reaction was observed in 117 of the 5,954 injections (2%) during the rapid phase and in 10 of the 5,567 injections (0.2%) during the maintenance phase, and all episodes disappeared immediately. Symptom score significantly decreased after SCIT, the effects being similar even after 3 years of cessation of SCIT. CONCLUSION: SCIT was effective for allergic rhinitis in childhood. The effect lasted after treatment cessation for a long term and the patient satisfaction was very high. There are some advantages of SCIT that sublingual immunotherapy does not have. SCIT should be considered as a choice for the treatment of allergic rhinitis in childhood.

[EVALUATION OF THE EFFECTIVENESS OF SUBCUTANEOUS IMMUNOTHERAPY USING BIRCH POLLEN EXTRACT FOR POLLEN-FOOD ALLERGY SYNDROME].

BACKGROUNDS: Recently, patients with oral allergy syndrome (OAS) to fruits or vegetables caused by pollenfood allergy syndrome (PFAS) have increased nationwide. We examined effectiveness of SCIT using birch pollen extract for PFAS. METHODS: A total of 19 patients (9 male and 10 female) underwent SCIT with birch pollen extract from August 2011 to August 2016. Rush schedule was used for the initial updosing for SCIT in a hospital setting. In maintenance phase, SCIT was administered every 4-8 weeks on an outpatient basis. According to the situation of sensitization, patients underwent SCIT with other extracts at the same time. Oral food challenge (OFC) with fruits and vegetables was performed at baseline and after rush phase. We also investigated about OAS symptoms in maintenance phase. RESULTS: SCIT was remarkably effective in five patients for OAS symptoms just after rush phase, and effective in nine patients. And it was not effective in two patients, and not determined in three patients, but it was confirmed to be effective in four out of these five patients in maintenance phase. There were relapse of OAS symptoms in three patients, then SCIT was remarkably effective or effective in 15 patients (79%) in maintenance phase. No patients dropped off the SCIT protocol. CONCLUSIONS: Generally, PFAS can't be expected to remit naturally. SCIT with birch pollen extract effectively reduces OAS symptoms, and it can be expected as a radical therapy for PFAS.

Genome-wide analysis of rice cis-natural antisense transcription under cadmium exposure using strand-specific RNA-Seq.

BACKGROUND: The elucidation of novel transcripts and their expression in response to various stress conditions is necessary to understand the transcriptional network of plants as an adaptation to biotic and abiotic stresses. We performed strand-specific RNA-Seq (ssRNA-Seq) on rice exposed to cadmium (Cd) for 24 h and investigated the expression of cis-natural antisense transcripts (cis-NATs), a class of endogenous coding or non-protein-coding RNAs with sequence complementarity to the opposite strands of RAP transcripts. RESULTS: Many RAP transcripts possessed cis-NATs and these cis-NATs were responsive to some extent. Cis-NATs were upregulated from 26, 266 and 409 RAP gene loci, while 2054, 2501 and 2825 RAP transcripts were upregulated from 38,123 RAP loci under high Cd exposure in roots at 1, 12 and 24 h, respectively. In addition, most of the upregulated cis-NATs showed little upregulation under ABA or cold treatment. A number of cis-NATs were upregulated from less than 35 RAP gene loci in different tissue and time-point combinations under low Cd exposure, suggesting that cis-NATs respond to environmental stress. Furthermore, 409 RAP transcripts with upregulated cis-NATs were classified into three groups based on the expression of the RAP transcripts from the opposite DNA strand, including 138 upregulated, 128 invariable, and 143 downregulated transcripts, although the responses of cis-NATs and RAP transcripts were not always correlated. CONCLUSIONS: We have shown that the cis-NATs identified by ssRNA-Seq analysis are novel genes and that some of them are stress-specific and show different responses depending on the degree of stress and tissue. These results improve our understanding of the complete molecular mechanism of plant adaptation to Cd exposure.

THE CASE OF A 14-YEAR-OLD BOY WHO EXPERIENCED ANAPHYLAXIS DUE TO JELLYFISH (MASTIGIAS PAPUA) INGESTION.

We report a case of jellyfish allergy diagnosed via an oral food challenge. A 14-year-old boy had no history of jellyfish stings and had been eating commercially available jellyfish products twice yearly for the past 5-6 years. Five minutes after eating a commercially available boiled jellyfish product (100g), he experienced nausea, wheezing, and erythema and had visited our hospital. We suspected an anaphylactic reaction and treated him with intramuscular adrenaline injection, corticosteroid and antihistamine infusions, volume resuscitation, and salbutamol sulfate inhalation, which resulted in an improvement of the symptoms. One-month later in our hospital, we administered an oral food challenge of the same boiled jellyfish product bought at the same grocery store to the patient. After ingesting 14g of boiled jellyfish, he experienced erythema, wheezing, nausea, and abdominal pain. Several reports have described anaphylaxis caused by the ingestion of jellyfish, but the allergens in jellyfish have not been analyzed. A skin prick test for poly-gamma-glutamic acid (PGA) which is a component of jellyfish stings was negative. This suggests that he was sensitized to some allergen other than PGA via a route different from that of jellyfish sting. Our skin prick test for several kinds of edible jellyfish suggests that allergenicity may be different for different jellyfish.

TENOR: Database for Comprehensive mRNA-Seq Experiments in Rice.

Here we present TENOR (Transcriptome ENcyclopedia Of Rice, http://tenor.dna.affrc.go.jp), a database that encompasses large-scale mRNA sequencing (mRNA-Seq) data obtained from rice under a wide variety of conditions. Since the elucidation of the ability of plants to adapt to various growing conditions is a key issue in plant sciences, it is of great interest to understand the regulatory networks of genes responsible for environmental changes. We used mRNA-Seq and performed a time-course transcriptome analysis of rice, Oryza sativa L. (cv. Nipponbare), under 10 abiotic stress conditions (high salinity; high and low phosphate; high, low and extremely low cadmium; drought; osmotic; cold; and flood) and two plant hormone treatment conditions (ABA and jasmonic acid). A large number of genes that were responsive to abiotic stresses and plant hormones were detected by differential expression analysis. Furthermore, several responsive genes were found to encode transcription factors that could control the transcriptional network of stress responses, but the timing of the induction of these genes was not uniform across conditions. A significant number of cis-regulatory elements were enriched in the promoter regions of the responsive genes and were shared among conditions. These data suggest that some key components of gene regulation networks are shared between different stress signaling pathways. All the resources (novel genes identified from mRNA-Seq data, expression profiles, co-expressed genes and cis-regulatory elements) can be searched for and are available in TENOR.

Genomic dissection of a 'Fuji' apple cultivar: re-sequencing, SNP marker development, definition of haplotypes, and QTL detection.

'Fuji' is one of the most popular and highly-produced apple cultivars worldwide, and has been frequently used in breeding programs. The development of genotypic markers for the preferable phenotypes of 'Fuji' is required. Here, we aimed to define the haplotypes of 'Fuji' and find associations between haplotypes and phenotypes of five traits (harvest day, fruit weight, acidity, degree of watercore, and flesh mealiness) by using 115 accessions related to 'Fuji'. Through the re-sequencing of 'Fuji' genome, total of 2,820,759 variants, including single nucleotide polymorphisms (SNPs) and insertions or deletions (indels) were detected between 'Fuji' and 'Golden Delicious' reference genome. We selected mapping-validated 1,014 SNPs, most of which were heterozygous in 'Fuji' and capable of distinguishing alleles inherited from the parents of 'Fuji' (i.e., 'Ralls Janet' and 'Delicious'). We used these SNPs to define the haplotypes of 'Fuji' and trace their inheritance in relatives, which were shown to have an average of 27% of 'Fuji' genome. Analysis of variance (ANOVA) based on 'Fuji' haplotypes identified one quantitative trait loci (QTL) each for harvest time, acidity, degree of watercore, and mealiness. A haplotype from 'Delicious' chr14 was considered to dominantly cause watercore, and one from 'Ralls Janet' chr1 was related to low-mealiness.

Incidence and factors related to delirium in an intensive care unit.

OBJECTIVES: To identify the incidence of delirium, compare the demographic and clinical characteristics of patients with and without delirium, and verify factors related to delirium in critical care patients. METHOD: Prospective cohort with a sample made up of patients hospitalized in the Intensive Care Unit (ICU) of a university hospital. Demographic, clinical variables and evaluation with the Confusion Assessment Method for Intensive Care Unit to identify delirium were processed to the univariate analysis and logistic regression to identify factors related to the occurrence of delirium. RESULTS: Of the total 149 patients in the sample, 69 (46.3%) presented delirium during ICU stay, whose mean age, severity of illness and length of ICU stay were statistically higher. The factors related to delirium were: age, midazolam, morphine and propofol. CONCLUSION: Results showed high incidence of ICU delirium associated with older age, use of sedatives and analgesics, emphasizing the need for relevant nursing care to prevent and identify early, patients presenting these characteristics. OBJETIVOS: Identificar a incidência de delirium, comparar as características demográficas e clínicas dos pacientes com e sem delirium e verificar os fatores relacionados ao delirium em pacientes internados em Unidade de Terapia Intensiva (UTI). MÉTODO: Coorte prospectiva, cuja amostra foi constituída de pacientes internados em UTI de um hospital universitário. Variáveis demográficas, clínicas e da avaliação com o Confusion Assessment Method for Intensive Care Unit para identificação de delirium foram processadas para análise univariada, e regressão logística para identificar fatores relacionados à ocorrência do delirium. RESULTADOS: Do total de 149 pacientes da amostra, 69 (46,3%) apresentaram delirium durante a internação na UTI, observando-se que a média da idade, o índice de gravidade e o tempo de permanência nas UTI foram estatisticamente maiores. Os fatores relacionados ao delirium foram: idade, midazolam, morfina e propofol. CONCLUSÃO: Os resultados mostraram elevada incidência de delirium na UTI e sua ocorrência associada às idades mais avançadas e o uso de sedativos e analgésicos, ressaltando-se a importância da atuação do enfermeiro na prevenção e identificação precoce do quadro nos pacientes com essas características.

Construction of a high-density mutant library in soybean and development of a mutant retrieval method using amplicon sequencing.

BACKGROUND: Functions of most genes predicted in the soybean genome have not been clarified. A mutant library with a high mutation density would be helpful for functional studies and for identification of novel alleles useful for breeding. Development of cost-effective and high-throughput protocols using next generation sequencing (NGS) technologies is expected to simplify the retrieval of mutants with mutations in genes of interest. RESULTS: To increase the mutation density, seeds of the Japanese elite soybean cultivar Enrei were treated with the chemical mutagen ethyl methanesulfonate (EMS); M2 seeds produced by M1 plants were treated with EMS once again. The resultant library, which consisted of DNA and seeds from 1536 plants, revealed large morphological and physiological variations. Based on whole-genome re-sequencing analysis of 12 mutant lines, the average number of base changes was 12,796 per line. On average, 691 and 35 per line were missense and nonsense mutations, respectively. Two screening strategies for high resolution melting (HRM) analysis and indexed amplicon sequencing were designed to retrieve the mutants; the mutations were confirmed by Sanger sequencing as the final step. In comparison with HRM screening of several genes, indexed amplicon sequencing allows one to scan a longer sequence range and skip screening steps and to know the sequence information of mutation because it uses systematic DNA pooling and the index of NGS reads, which simplifies the discovery of mutants with amino acid substitutions. CONCLUSIONS: A soybean mutant library with a high mutation density was developed. A high mutation density (1 mutation/74 kb) was achieved by repeating the EMS treatment. The mutation density of our library is sufficiently high to obtain a plant in which a gene is nonsense mutated. Thus, our mutant library and the indexed amplicon sequencing will be useful for functional studies of soybean genes and have a potential to yield useful mutant alleles for soybean breeding.

A BAC physical map of aus rice cultivar 'Kasalath', and the map-based genomic sequence of 'Kasalath' chromosome 1.

Comparative analysis using available genomic resources within closely related species is an effective way to investigate genomic sequence and structural diversity. Rice (Oryza sativa L.) has undergone significant physiological and morphological changes during its domestication and local adaptation. We present a complete bacterial artificial chromosome (BAC) physical map for the aus rice cultivar 'Kasalath', which covers 90% of the sequence of temperate japonica rice cultivar 'Nipponbare'. Examination of physical distances between computational and experimental measurements of 'Kasalath' BAC insert size revealed the presence of more than 500 genomic regions that appear to have significant chromosome structural changes between the two cultivars. In particular, a genomic region on the long arm of 'Kasalath' chromosome 11 carrying a disease-resistance gene cluster was greatly expanded relative to the 'Nipponbare' genome. We also decoded 41.37 Mb of high-quality genomic sequence from 'Kasalath' chromosome 1. Extensive comparisons of chromosome 1 between 'Kasalath' and 'Nipponbare' led to the discovery of 317,843 single-nucleotide polymorphisms (SNPs) and 66,331 insertion/deletion (indel) sites. Nearly two-thirds of the expressed genes on rice chromosome 1 carried natural variations involving SNPs and/or indels that resulted in substitutions, insertions or deletions of amino acids in one cultivar relative to the other. We also observed gain and loss of genes caused by large indels. This study provides an important framework and an invaluable dataset for further understanding of the molecular mechanisms underlying the evolution and functions of the rice genome.

Electrokinetic supercharging preconcentration prior to CGE analysis of DNA: sensitivity depends on buffer viscosity and electrode configuration.

Aiming to high sensitivity DNA analysis by CGE, electrokinetic supercharging (EKS) approach was adopted in this article. EKS is known as an online preconcentration technique that combines electrokinetic sample injection (EKI) with transient ITP (tITP). Herein, two factors of buffer viscosity and electrode configuration were studied to further improve EKS performance. An ultralow-viscosity Tris-Boric acid-EDTA (TBE) buffer solution, consisted of 2% low-molecular-weight hydroxypropyl methyl cellulose (HPMC) and 6% mannitol and with pH 8.0 adjusted by boric acid, was applied. The boric acid would make a complex with mannitol and generates borate polyanion, which acts as the leading ion for tITP process. The new electrode configuration, a Pt ring around capillary, was modified on Agilent CE system to lead large amount sample introduction during EKS. The standard DNA sample of φX174/HaeIII digest was used to evaluate the qualitative and quantitative abilities of the proposed strategy. The 170,000-fold highly diluted sample at concentration of 3.0 ng/mL was enriched by EKS and detected by normal UV detection method. The obtained LOD of the weakest peak of 72 bp fragment was around 7.7 pg/mL, apparently improved more than 10,000-fold in comparison with conventional CGE with UV detection.

DNA aggregation and cleavage in CGE induced by high electric field in aqueous solution accompanying electrokinetic sample injection.

The phenomenon of peak area decrease due to high injection voltage (Vinj , e.g. 10-30 kV, 200-600 V/cm in the 50 cm capillary) was found in the analysis of very dilute DNA fragments (<0.2 mg/L) by using high-sensitive electrokinetic supercharging-CGE. The possibility of DNA cleavage in aqueous solution was suggested, in addition to the aggregation phenomenon that is already known. The analysis of intentionally voltage-affected fragments (at 200 V/cm) also showed decreased peak areas depending on the time of the voltage being applied. Computer simulation suggested that a high electric field (a few kV/cm or more) could be generated partly between the electrode and the capillary end during electrokinetic injection (EKI) process. After thorough experimental verification, it was found that the factors affecting the damage during EKI were the magnitude of electric field, the distance between tips of electrode and capillary (De/c ), sample concentration and traveling time during EKI in sample vials. Furthermore, these factors are correlating with each other. A low conductivity of diluted sample would cause a high electric field (over a few hundred volts per centimeter), while the longer De/c results in a longer traveling time during EKI, which may cause a larger degree of damage (aggregation and cleavage) on the DNA fragments. As an important practical implication of this study, when the dilute DNA fragments (sub mg/L) are to be analyzed by CGE using EKI, injection voltage should be kept as low as possible.

[Evaluation of an educational website on First Aid]. / Avaliação do website educacional em Primeiros Socorros.

The aim of this study was to evaluate the structure, quality of information and usability of a website on First Aid. The evaluation was performed by information technology (IT) and health care professionals and by students, using specific and validated instruments. The kappa method was used to evaluate the agreement of the answers, and Cronbach's α coefficient was used to assess the reliability of the instrument. There was no agreement (0.047) among the answers obtained from the IT professionals, indicating that the structure of the website must be reviewed. There was also no agreement in the evaluation by the health care professionals (-0.062); however, the overall positive scores suggest that the quality of the information of the website is adequate. The assessment of reliability of the instrument to evaluate the navigability rendered a value of α=0.974. Although improvement of the website structure is recommended, the quality of the information is good, and its use has contributed to the apprenticeship of students.

Case of a 6-year-old boy with anaphylaxis induced by erythritol with positive skin prick test and negative basophil activation test.

Erythritol is widely used as an additive in foods and pharmaceuticals. We present the case of a 6-year-old boy who developed an allergy to erythritol. He showed a positive skin prick test result and a negative basophil activation test result. In cases involving allergens with low molecular weights, the test results should be carefully interpreted.

Factors associated with the use of physical restraint in intensive care patients.

OBJECTIVE: To verify the frequency of physical restraint in patients and the factors associated with its use in the intensive care unit. METHOD: An observational and prospective study on the use of restraint in patients observed over two days, considering the variables: age and gender, personal and clinical characteristics, devices, adverse event and restraint use. The frequency was verified in three groups of patients with different conditions by applying the Chi-Squared, Likelihood Ratio or Kruskal-Wallis tests. The association of the variables was verified with the Multinomial Logistic Regression. RESULTS: Eighty-four (84) patients participated. Restraint was observed in 77.4% of the 84 analyzed patients, and was more frequent in the presence of sedation, agitation and invasive devices. The chance of being restrained was at least five times higher in sedation conditions, whether in weaning or daily awakening, mechanical ventilation weaning, agitation or the presence of invasive devices. CONCLUSION: Restraint use was high and was associated with female gender, sedation, agitation and invasive airway. It is emphasized and important to apply policies to reduce restraint use in intensive care.

Photoprotective Effects of Selected Amino Acids on Naproxen Photodegradation in Aqueous Media.

It is important to develop a photostabilization strategy to ensure the quality of photosensitive compounds, including pharmaceuticals. This study focused on the protective effects of 20 amino acids on the photodegradation of naproxen (NX), a photosensitive pharmaceutical, to clarify the important nature of a good photostabilizer. Our previous report indicated the photodegradability of NX and the protective effects of some antioxidants on its photodegradation, therefore, this compound was used as a model compound. The degradation of NX in aqueous media during ultraviolet light (UV) irradiation and the protective effects of selected amino acids were monitored through high-performance liquid chromatography (HPLC), equipped with a reverse-phase column. Addition of cysteine, tryptophan, and tyrosine induced the significant suppression of NX photodegradation after UV irradiation for 3 h (residual amount of NX; 15.35%, 6.82%, and 15.64%, respectively). Evaluation of the antioxidative activity and UV absorption spectrum showed that cysteine suppressed NX degradation through its antioxidative ability, while tryptophan and tyrosine suppressed it through their UV filtering ability. Furthermore, three amino acids at higher concentrations (more than 100 µmol/L) showed more protective effects on NX photodegradation. For 10 mmol/L, residual amounts of NX with cysteine, tryptophan, and tyrosine were 58.51%, 69.34%, and 82.40%, respectively. These results showed the importance of both photoprotective potencies (antioxidative potency and UV filtering potency) and stability to UV irradiation for a good photostabilizer of photosensitive pharmaceuticals.

Metagenomic profiles of the rumen microbiota during the transition period in low-yield and high-yield dairy cows.

We investigated potential relationships between rumen microbiota and milk production in dairy cows during the transition period. Twelve dairy cows were divided into a low-yield (LY) or high-yield (HY) group based on their milk yield. Rumen samples were taken from dairy cows at 3 weeks before parturition, and at 4, 8, and 12 weeks after parturition. 16S rDNA-based metagenomic analysis showed that diversities of rumen microbiota in both groups were similar and the number of operational taxonomic units (OTUs) was lower in the postpartum than prepartum period in both groups. The abundance of Bacteroidetes and ratio of Bacteroidetes:Firmicutes was higher in the HY than the LY group. OTUs assigned to Prevotella bryantii, Fibrobacter succinogenes, Ruminococcus albus, Butyrivibrio fibrisolvens, and Succinivibrio sp. were abundant in the HY group. These OTUs were significantly related to the propionate molar proportion of rumen fluids in the HY group. OTUs assigned to Lachnospiraceae, Bifidobacterium sp. and Saccharofermentans were dominant in the LY group. Predictive functional profiling revealed that abundance of gene families involved in amino acid and vitamin metabolism was higher in the HY than the LY group. These results suggest that the community structure and fermentation products of rumen microbiota could be associated with milk production of dairy cows.

Genome-Wide Transcriptome Analysis of Cadmium Stress in Rice.

Rice growth is severely affected by toxic concentrations of the nonessential heavy metal cadmium (Cd). To elucidate the molecular basis of the response to Cd stress, we performed mRNA sequencing of rice following our previous study on exposure to high concentrations of Cd (Oono et al., 2014). In this study, rice plants were hydroponically treated with low concentrations of Cd and approximately 211 million sequence reads were mapped onto the IRGSP-1.0 reference rice genome sequence. Many genes, including some identified under high Cd concentration exposure in our previous study, were found to be responsive to low Cd exposure, with an average of about 11,000 transcripts from each condition. However, genes expressed constitutively across the developmental course responded only slightly to low Cd concentrations, in contrast to their clear response to high Cd concentration, which causes fatal damage to rice seedlings according to phenotypic changes. The expression of metal ion transporter genes tended to correlate with Cd concentration, suggesting the potential of the RNA-Seq strategy to reveal novel Cd-responsive transporters by analyzing gene expression under different Cd concentrations. This study could help to develop novel strategies for improving tolerance to Cd exposure in rice and other cereal crops.