Comparison of endoscopic submucosal resection with ligation and endoscopic submucosal dissection for small rectal neuroendocrine tumors: A multicenter retrospective study.

Objectives: Endoscopic submucosal resection with band ligation (ESMR-L) and endoscopic submucosal dissection (ESD) are both standard endoscopic resection methods for rectal neuroendocrine tumors (NETs) <10 mm in size. However, there is no definitive consensus on which is better. Here, we compared the efficacy of ESMR-L and ESD for small rectal NETs. Methods: This was a multicenter retrospective cohort study including 205 patients with rectal NETs who underwent ESMR-L or ESD. Treatment outcomes were compared by univariate analysis, multivariate analysis, and inverse probability treatment weighting (IPTW) using propensity scores. Subgroup analysis evaluated the impact of the endoscopist's experience on the technical outcome. Results: Eighty-nine patients were treated by ESMR-L and 116 by ESD. The R0 resection rate was not significantly different between the two (90% vs. 92%, p = 0.73). The procedure time of ESMR-L was significantly shorter than for ESD (17 min vs. 52 min, p < 0.01) and the hospitalization period was also significantly shorter (3 days vs. 5 days, p < 0.01). These results were confirmed by multivariate analysis and also after IPTW adjustment. The procedure time of ESD was significantly prolonged by a less-experienced endoscopist (49 min vs. 70 min, p = 0.02), but that of ESMR-L was not affected (17 min vs. 17 min, p = 0.27). Conclusions: For small rectal NETs, both ESMR-L and ESD showed similar high complete resection rates. However, considering the shorter procedure time and shorter hospitalization period, ESMR-L is the more efficient treatment method, especially for less-experienced endoscopists.

Multicenter retrospective study of 132 patients with unresectable small bowel adenocarcinoma treated with chemotherapy.

BACKGROUND: No standard chemotherapy regimen has been established for unresectable or recurrent small bowel adenocarcinoma (SBA). METHODS: Clinical courses of 132 patients with unresectable or recurrent SBA who received chemotherapy at 41 institutions in Japan were reviewed retrospectively. Patients were classified into five groups according to first-line chemotherapy regimens: fluoropyrimidine monotherapy (group A), fluoropyrimidine-cisplatin (group B), fluoropyrimidine-oxaliplatin (group C), fluoropyrimidine-irinotecan (group D), and other regimens (group E). RESULTS: The number of patients in each group was as follows: groups A, 60 patients; group B, 17 patients; group C, 22 patients; group D, 11 patients; and group E, 22 patients. Median progression-free survival (PFS) times were as follows: group A, 5.4 months; group B, 3.8 months; group C, 8.2 months; group D, 5.6 months; and group E, 3.4 months. Median overall survival (OS) times were as follows: group A, 13.9 months; group B, 12.6 months; group C, 22.2 months; group D, 9.4 months; and group D, 8.1 months. Patients in group C achieved significantly longer PFS times and substantially (but not significantly) longer OS times than patients in group A. After adjusting for clinical background characteristics, fluoropyrimidine-oxaliplatin therapy was a significant positive prognostic factor for PFS and OS times. CONCLUSION: The results suggest that fluoropyrimidine-oxaliplatin combination therapy is the most promising first-line chemotherapy regimen for unresectable or recurrent SBA.

Draft genome sequence of Taiwanese pear (Pyrus pyrifolia).

Pear (genus Pyrus) is one of the oldest temperate fruit crops, of which at least 22 primary species are recognized. This article documents the public availability of the partial draft genome sequence data of the Taiwanese pear 'Hengshanli' that is less dormant during the winter season. This dataset may be used to prepare molecular markers for the breeding of new cultivars that are subjected to chilling at low temperatures to overcome endodormancy. This data will also help analyze the process of evolution in the Pyrus species. We sequenced paired-end libraries using Illumina HiSeq. 2500 and generated approximately 210M reads. Data on the draft genome obtained in this study has been deposited to the DNA Data Bank of Japan (DDBJ). The read data were submitted to the DDBJ Read Archive (BioProject: PRJDB6877, BioSample: SAMD00117052).

Draft genome sequence of Japanese pear (Pyrus pyrifolia).

The Japanese pear (Pyrus pyrifolia), a member of the family Rosaceae, is one of the most important fruit trees in Japan. This article documents the public availability of the partial draft genome sequence data of the Japanese pear strain TH3, which is the S1 of 'Osa-Nijisseiki' and is homozygous for the S4sm gene. This dataset may be used to prepare molecular markers for breeding of new cultivars having a crisp texture and feel. This data will also help research on physiological disorders affecting Japanese pear fruit. We sequenced paired-end libraries using Illumina HiSeq. 2500 and generated approximately 212M reads. Data on the draft genome obtained in this study has been deposited to the DNA Data Bank of Japan (DDBJ). The read data were submitted to the DDBJ Read Archive (BioProject: PRJDB6878, BioSample: SAMD00117051).

Identification of the expressed protein and the impact of change in ascorbate peroxidase activity related to endodormancy breaking in Pyrus pyrifolia.

Endodormancy is an important feature of perennial deciduous fruit trees that survive in the extreme climates brought about by seasonal variation. To acquire a comprehensive knowledge of the biochemical processes occurring just before endodormancy breaking, the buds collected in the pre-breaking period (PP) phase were used as samples to identify the proteins related to the breaking of endodormancy in the Japanese pear (Pyrus pyrifolia Nakai). Using nano-ESI-LC-MS/MS analysis, 96 proteins were overlapped by analyses of three times and identified as expressed proteins at the PP stage. Among these proteins, dehydrin, several classes of heat shock proteins (HSP), auxin-binding protein, and auxin-induced protein were identified in the floral bud in the PP stage. The majority of these proteins were involved primarily in the oxidation-reduction process. We focused on catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX) as enzymes regulating the levels of hydrogen peroxide (H2O2) in the bud. From measurements taken during the deepest period (DP), PP, mid-breaking period (MP), and late-breaking period (LP) of endodormancy, CAT activity decreased gradually, while APX activity also decreased from DP to MP, but then increased rapidly during LP. Protein data for PP and the rapid increase in APX activity observed in LP provided knowledge of the biochemical processes that regulate the consecutive transition from endodormancy breaking to ecodormancy induction in the Japanese pear.

Gene expression changes triggered by end-of-day far-red light treatment on early developmental stages of Eustoma grandiflorum (Raf.) Shinn.

To better understand the molecular mechanisms related to growth promotion in the early developmental stages of Eustoma grandiflorum (Raf.) Shinn. under end-of-day far-red light (EOD-FR) treatment, we analyzed the leaf transcriptome of treated (EOD) and untreated plants (Cont) by using RNA-seq technology. EOD-FR treatment for only about 2 weeks in regions with limited sunshine during winter resulted in significantly higher internode length between the 3rd and 4th nodes on the main stem in EOD than in Cont. Among the differentially expressed genes (DEGs) related to synthesis or transport of auxin, higher levels of YUCCA (CL6581) and PIN4 (CL6181) were noted after treatment in EOD than in Cont in the leaf. In addition, high expression levels of GA20ox (Unigene11862) related to gibberellin (GA) synthesis and transcription factor bHLH 135 (CL7761) were observed in the stem of EOD, 3 h after treatment. A vertical section of the stem showed that the pith length of cells at the 4th node was longer in EOD than in Cont. Collectively, these results suggested that EOD-FR treatment increased the expression of DEGs related to GA and auxin biosynthesis, bHLH transcription factor, and internodal cell elongation along the longitudinal axis of Eustoma plants.

Comparative Transcriptome Analysis of the Less-Dormant Taiwanese Pear and the Dormant Japanese Pear during Winter Season.

The flower bud transcriptome in the less dormant Taiwanese pear 'Hengshanli' and high-chilling requiring Japanese pear strain TH3 subjected to the same chilling exposure time were analyzed during winter using next-generation sequencing. In buds sampled on January 10th and on February 7th in 2014, 6,978 and 7,096 genes, respectively, were significantly differentially expressed in the TH3 and 'Hengshanli' libraries. A comparative GO analysis revealed that oxidation-reduction process (biological process) and ATP binding (molecular function), were overrepresented during the ecodormancy period (EP) when compared to the endodormancy deepest period (DP), indicating that ATP synthesis was activated during the transition between these dormancy stages. Among the 11 differently expressed genes (DEGs) annotated as probable dehydrins or LEA protein-related genes, 9 DEGs showed higher transcript levels in the DP than in the EP. In order to focus on transcription factors induced by low temperature or drought, 7 differently expressed genes (DEGs) annotated as probable ICE1 or DREB proteins were analyzed by real-time PCR. Expression levels of 3 genes were higher in TH3 than in 'Hengshanli' on all sampling days. Their expression increased during the endodormancy deepest period (DP) and then decreased before endodormancy breaking in TH3 buds. Taken together, these results suggest that these genes annotated as ICE1, DREB and ERF are involved in endodormancy maintenance and in the transition from endodormancy to ecodormancy.

Relationship between nutrition status and dental occlusion in community-dwelling frail elderly people.

AIM: This study aimed to determine the risk of malnutrition in some communities where the frail elderly receive public long-term care insurance. We also clarified the dental problems in those at risk of malnutrition. METHODS: A total of 716 frail elderly who lived in eight cities in Japan (240 males and 476 females with a mean age of 83.2±8.6 years) were divided into three groups according to Mini Nutritional Assessment short form results: well nourished, at risk of malnutrition and malnourished. They were also divided into three groups in terms of remaining teeth occlusion and denture occlusion: group A, natural dentition with adequate function; group B, partially or fully edentulous, but maintaining functional occlusion with dentures in either or both jaws; and group C, functionally inadequate occlusion with no dentures. The relationship between nutrition status and dental occlusion was evaluated using logistic regression analysis with sex, age, activities of daily living and cognitive function as covariates. RESULTS: The number of participants in each of the groups was as follows: 251 well nourished, 370 at risk of malnutrition and 95 malnourished. When they were divided into just two groups, (i) well nourished and (ii) at risk of malnutrition plus malnourished, in order to study malnutrition risk factors, there were significant relationships between their nutritious status and sex, Barthel index, and occlusion. CONCLUSION: This large-scale cross-sectional survey showed that loss of natural teeth occlusion was a risk factor for malnutrition among community-dwelling frail elderly.

Identification of the major antigenic protein of Helicobacter cinaedi and its immunogenicity in humans with H. cinaedi infections.

Helicobacter cinaedi infection is now recognized as an increasingly important emerging disease. Its pathogenesis and epidemiological features are not fully understood, however. Here, we investigated the antigenic protein of H. cinaedi and the immunological response to it in H. cinaedi-infected patients. We constructed a genomic library of H. cinaedi from an H. cinaedi clinical isolate, and various H. cinaedi recombinant proteins were expressed. We identified the 30-kDa protein, encoded in an 822-bp H. cinaedi genome, as a major antigen, which was specifically recognized by serum from an H. cinaedi-immunized rabbit and H. cinaedi-infected patients. The gene encoding this 30-kDa antigen had high sequence similarity with genes encoding putative membrane proteins of bacteria. To evaluate whether the 30-kDa protein can be applied in serological testing for H. cinaedi infections, the recombinant protein was expressed in Escherichia coli as a His-tagged fusion protein and purified by Ni(2+) affinity chromatography. Western blot analysis revealed strong immunoreactivity of the 31-kDa fusion protein with serum antibody from patients infected with H. cinaedi, but such an immunoreaction was absent or was very weak with uninfected control serum. An enzyme-linked immunosorbent assay using this H. cinaedi major antigen showed significantly high antibody titers for H. cinaedi-infected subjects compared with those of various control groups. We therefore conclude that the 30-kDa putative membrane protein is a major antigen of H. cinaedi and is useful for immunological and serological testing for clinical diagnosis and for further epidemiological study of H. cinaedi infection in humans.

The impact of cell division and cell enlargement on the evolution of fruit size in Pyrus pyrifolia.

BACKGROUND AND AIMS: Dramatic increases in fruit size have accompanied the domestication of Pyrus pyrifolia. To evaluate the contribution of cell division and cell enlargement in the evolution of fruit size, the following study was conducted. METHODS: Three wild Pyrus and 46 cultivated Pyrus pyrifolia cultivars were selected to examine cell number/size at time of pollination and at time of fruit harvest. The period of cell division was estimated by logarithmic curve of the increasing pattern of cell number, and its correlations with maturation period and final fruit size were analysed. KEY RESULTS: Final fruit size is directly related to the number of cells produced in the period immediately following pollination. Late-maturing cultivars are larger than earlier-maturing cultivars and this is due to an extended period of cell division. CONCLUSIONS: The evolution of fruit size in P. pyrifolia has mainly resulted from shifts in the ability of cells to divide rather than to enlarge.

Partitioning of (13)C-photosynthate from spur leaves during fruit growth of three Japanese pear (Pyrus pyrifolia) cultivars differing in maturation date.

BACKGROUND AND AIMS: In fruit crops, fruit size at harvest is an important aspect of quality. With Japanese pears (Pyrus pyrifolia), later maturing cultivars usually have larger fruits than earlier maturing cultivars. It is considered that the supply of photosynthate during fruit development is a critical determinant of size. To assess the interaction of assimilate supply and early/late maturity of cultivars and its effect on final fruit size, the pattern of carbon assimilate partitioning from spur leaves (source) to fruit and other organs (sinks) during fruit growth was investigated using three genotypes differing in maturation date. METHODS: Partitioning of photosynthate from spur leaves during fruit growth was investigated by exposure of spurs to (13)CO(2) and measurement of the change in (13)C abundance in dry matter with time. Leaf number and leaf area per spur, fresh fruit weight, cell number and cell size of the mesocarp were measured and used to model the development of the spur leaf and fruit. KEY RESULTS: Compared with the earlier-maturing cultivars 'Shinsui' and 'Kousui', the larger-fruited, later-maturing cultivar 'Shinsetsu' had a greater total leaf area per spur, greater source strength (source weight x source specific activity), with more (13)C assimilated per spur and allocated to fruit, smaller loss of (13)C in respiration and export over the season, and longer duration of cell division and enlargement. Histology shows that cultivar differences in final fruit size were mainly attributable to the number of cells in the mesocarp. CONCLUSIONS: Assimilate availability during the period of cell division was crucial for early fruit growth and closely correlated with final fruit size. Early fruit growth of the earlier-maturing cultivars, but not the later-maturing ones, was severely restrained by assimilate supply rather than by sink limitation.

13C-photosynthate accumulation in Japanese pear fruit during the period of rapid fruit growth is limited by the sink strength of fruit rather than by the transport capacity of the pedicel.

In Japanese pear, the application of GA3+4 during the period of rapid fruit growth resulted in a marked increase in pedicel diameter and bigger fruit at harvest. To elucidate the relationship between pedicel capacity and fruit growth and to determine the main factor responsible for larger fruit size at harvest, fruit growth and pedicel vascularization after GA application were examined and the carbohydrate fluxes were monitored in a spur unit by non-invasive techniques using 13C tracer. Histological studies of fruit revealed that GA increased the cell size of the mesocarp but not the cell number and core size. The investigation of carbon partitioning showed that an increase in the specific rate of carbohydrate accumulation in fruit or the strength of fruit should be responsible for an increase of fruit weight in GA-treated trees. Observation of pedicel vascularization showed that an increase in pedicel cross-sectional area (CSA) by GA application mainly resulted from phloem and xylem CSA, but it is unlikely that an increase in the transport system is the direct factor for larger fruit size. Therefore, it can be concluded that larger fruit size resulting from GA application during the period of rapid fruit growth caused an increase in the cell size of the mesocarp and increased carbon partitioning to the fruit. Although GA is closely involved with pedicel vascularization, it seems that photosynthate accumulation in fruit is limited by the sink strength of fruit rather than by the transport capacity of the pedicel.

Nitric oxide as an endogenous mutagen for Sendai virus without antiviral activity.

Nitric oxide (NO) may affect the genomes of various pathogens, and this mutagenesis is of particular interest for viral pathogenesis and evolution. Here, we investigated the effect of NO on viral replication and mutation. Exogenous or endogenous NO had no apparent antiviral effect on influenza A virus and Sendai virus. The mutagenic potential of NO was analyzed with Sendai virus fused to a green fluorescent protein (GFP) gene (GFP-SeV). GFP-SeV was cultured in SW480 cells transfected with a vector expressing inducible NO synthase (iNOS). The mutation frequency of GFP-SeV was examined by measuring loss of GFP fluorescence of the viral plaques. GFP-SeV mutation frequency in iNOS-SW480 cells was much higher than that in parent SW480 cells and was reduced to the level of mutation frequency in the parent cells by treatment with an NO synthase (NOS) inhibitor. Immunocytochemistry showed generation of more 8-nitroguanosine in iNOS-SW480 cells than in SW480 cells without iNOS transfection. Authentic 8-nitroguanosine added exogenously to GFP-SeV-infected CV-1 cells increased the viral mutation frequency. Profiles of the GFP gene mutations induced by 8-nitroguanosine appeared to resemble those of mutations occurring in mouse lungs in vivo. A base substitution that was characteristic of both mutants (those induced by 8-nitroguanosine and those occurring in vivo) was a C-to-U transition. NO-dependent oxidative stress in iNOS-SW480 cells was also evident. Together, the results indicate unambiguously that NO has mutagenic potential for RNA viruses such as Sendai virus without affecting viral replication, possibly via 8-nitroguanosine formation and cellular oxidative stress.

Molecular mechanism for activation and regulation of matrix metalloproteinases during bacterial infections and respiratory inflammation.

Matrix metalloproteinases (MMPs) are critical mediators of tissue remodeling. Inappropriate regulation of MMPs causes many pathological events, including microbial invasion and inflammatory tissue damage. Some of the bacterial exoproteinases can effectively activate pro-MMPs (inactive zymogens) via limited proteolysis around their autoinhibitory domains. In addition, overproduction of nitric oxide (NO) may contribute to respiratory inflammation via the formation of reactive nitrogen species (RNS). Several studies have identified regulatory properties of NO/RNS on biomolecules due to functional modification of their cysteine residues. In fact, NO/RNS can mediate activation and expression of MMPs, because RNS can interact with a cysteine switch in the autoinhibitory domain, thus converting proMMPs into their active forms without proteolysis. Many studies have indicated that NO/RNS can participate in expression of various genes that affect immune-inflammatory responses, including MMPs. Although NO in some cases upregulates MMPs, S -nitrosothiols downregulate MMP-9 expression by suppressing the NF-kappaB pathway. While microbial proteinases cause excessive activation of MMPs and contribute to microbial pathogenesis, NO/RNS may modulate expression and activation of MMPs as well as various inflammatory mediators, depending on the redox status at sites of inflammation. Therefore, appropriate regulation of MMPs may be of potential therapeutic value for various infections and inflammatory lung diseases.

8-nitroguanosine formation in viral pneumonia and its implication for pathogenesis.

For many diseases, mediation of pathogenesis by nitric oxide (NO) has been suggested. In this study, we explored NO-induced viral pathogenesis with a focus on nucleic acid damage as evidenced by 8-nitroguanosine formation in vivo. Wild-type mice and littermate mice deficient in inducible NO synthase (iNOS) were infected with influenza or Sendai virus. Formation of 8-nitroguanosine in virus-infected lungs was assessed immunohistochemically with an antibody specific for 8-nitroguanosine. Extensive nitration of RNA either treated with peroxynitrite or obtained from cultured RAW 264 cells expressing iNOS was readily detected by this antibody. Strong 8-nitroguanosine immunostaining was evident primarily in the cytosol of bronchial and bronchiolar epithelial cells of virus-infected wild-type mice but not iNOS-deficient mice. This staining colocalized with iNOS immunostaining in the lung. 8- Nitroguanosine staining disappeared after addition of exogenous authentic 8-nitroguanosine during the antibody reaction and after pretreatment of tissues with sodium hydrosulfite, which reduces 8-nitroguanosine to 8-aminoguanosine. NO was generated in excess in lungs of wild-type mice but was eliminated in iNOS-deficient mice after virus infection; this result also correlated well with formation of 8-nitroguanosine and 3-nitrotyrosine. One consequence of the lack of iNOS expression was marked improvement in histopathological changes in the lung and the lethality of the infection without effects on cytokine responses and viral clearance. It is intriguing that 8-nitroguanosine markedly stimulated superoxide generation from cytochrome P450 reductase and iNOS in vitro. The present data constitute a demonstration of 8-nitroguanosine formation in vivo and suggest a potential role for NO-induced nitrative stress in viral pathogenesis.

Proapoptotic effect of proteolytic activation of matrix metalloproteinases by Streptococcus pyogenes thiol proteinase (Streptococcus pyrogenic exotoxin B).

Streptococcus pyogenes thiol proteinase, also known as streptococcal pyrogenic exotoxin B (SpeB), has been suggested to be a major virulence factor in S. pyogenes infection. SpeB was reported to induce apoptosis of host cells, but its mechanism of action is not yet fully understood. In this study, we examined the involvement of matrix metalloproteinases (MMPs) in SpeB-induced apoptosis. We first developed a large-scale preparation of recombinant SpeB and precursors of human MMP-9 and -2 (proMMPs) by using Escherichia coli Rosetta (DE3)pLysS and baculovirus-insect cell expression systems, respectively. Treatment with SpeB induced effective proteolytic activation of both proMMP-9 and -2. When RAW264 murine macrophages were incubated with SpeB-activated proMMP-9, the level of tumor necrosis factor alpha (TNF-alpha) in conditioned medium (CM), assessed by an enzyme immunoassay, was elevated. This increase was completely inhibited by addition of the MMP inhibitor SI-27 to the cell culture. The CM also produced marked induction of apoptosis of U937 human monocytic cells. Similarly, soluble Fas ligand (sFasL) was detected in CM of cultures of SW480 cells expressing FasL after treatment with SpeB-activated proMMPs; this CM also induced apoptosis in U937 cells. SpeB had a direct effect as well and caused the release of TNF-alpha and sFasL from the cells. SpeB-dependent production of MMP-9 and -2 and proapoptotic molecules (TNF-alpha and sFasL) was evident in a murine model of severe invasive S. pyogenes infection. These results suggest that SpeB or SpeB-activated MMPs contribute to tissue damage and streptococcal invasion in the host via extracellular release of TNF-alpha and sFasL.

Role of nitric oxide in host defense in murine salmonellosis as a function of its antibacterial and antiapoptotic activities.

Host defense functions of nitric oxide (NO) are known for many bacterial infections. In this study, we investigated the antimicrobial effect of NO in murine salmonellosis by using inducible NO synthase (iNOS)-deficient mice infected with an avirulent or virulent Salmonella enterica serovar Typhimurium strain. All iNOS-deficient mice died of severe septicemia within 6 days after intraperitoneal injection with an avirulent strain (LT2) to which wild-type mice were highly resistant; 50% lethal doses (LD(50)s) of the LT2 strain for iNOS-deficient and wild-type mice were 30 CFU and 7 x 10(4) CFU, respectively. Lack of NO production in iNOS-deficient mice was verified directly by electron spin resonance spectroscopy. Bacterial yields in liver and blood were much higher in iNOS-deficient mice than in wild-type mice throughout the course of infection. Very small amounts of a virulent strain of serovar Typhimurium (a clinical isolate, strain Gifu 12142; LD(50), 50 CFU) given orally caused severe septicemia in iNOS-deficient animals; wild-type mice tolerated higher doses (LD(50), 6 x 10(2) CFU). Histopathology of livers from infected iNOS-deficient mice revealed extensive damage, such as diffuse hepatocellular apoptosis and increased neutrophil infiltration, but livers from infected wild-type mice showed a limited number of microabscesses, consisting of polymorphonuclear cells and macrophages and low levels of apoptotic change. The LT2 strain was much more susceptible to the bactericidal effect of peroxynitrite than the Gifu strain, suggesting that peroxynitrite resistance may contribute to Salmonella pathogenicity. These results indicate that NO has significant host defense functions in Salmonella infections not only because of its direct antimicrobial effect but also via cytoprotective actions for infected host cells, possibly through its antiapoptotic effect.