Faecal freezing preservation period influences colonization ability for faecal microbiota transplantation.

AIMS: There has been growing interest in faecal microbiota transplantation (FMT) as treatment. Although, frozen donor faeces preserved at -20°C has been widely used for practical advantages, freezing at -20°C can affect bacterial viability. Adequacy evaluation of fresh and frozen faeces as the transplant is necessary for the methodological improvement of FMT. METHODS AND RESULTS: The viable bacterial compositions of faecal specimens under fresh and freezing conditions were compared by a microbiome analysis using propidium monoazide (PMA microbiome). In addition, recovery abilities from bacterial reduction by antibiotics were compared between fresh and frozen FMT using a murine model. PMA microbiome results suggested that freezing and freeze-thawing did not significantly affect in vitro faecal bacterial viability. However, the recovery effect from antimicrobial cleansing in frozen FMT was reduced in a freezing time-dependent manner, especially prominent in Actinobacteria and Bacteroidetes phyla. CONCLUSIONS: Short-term freezing preservation of faeces exhibited maintenance of enteric colonization ability in frozen FMT in comparison to 1 month -20°C-preservation. SIGNIFICANCE AND IMPACT OF THE STUDY: Long-term -20°C-preservation of transplanted faeces can result in instability of the clinical outcome in FMT therapy. The standardization of practical procedures of FMT therapy according to disease types is desirable.

Atmospheric Humidity Influences Oviposition Rate of Tetranychus urticae (Acari: Tetranychidae) Through Morphological Responses of Host Cucumis sativus Leaves.

We investigated the effects of morphology of host cucumber, Cucumis sativus L., leaves acclimatized to different atmospheric humidity levels on oviposition by adult females of the twospotted spider mite, Tetranychus urticae Koch. Cucumber seedlings were grown at a vapor pressure deficit (VPD) of 0.4, 1.9, or 3.0 kPa at 28°C (90%, 50%, or 20% relative humidity, respectively) in growth chambers until the second true leaves had expanded. Adult females of T. urticae were released on the adaxial surfaces of leaf squares cut from first and second true leaves in each treatment group, and held in the same humidity condition. Eggs were counted 2 d after release. The lower acclimatization humidity (higher VPD) increased trichome (leaf hair) density of the host leaves and oviposition rate, but the relationship between the trichome and oviposition differed between leaf positions. The leaf mass per area (LMA) was greater in first true leaves than in second true leaves, but was not influenced by VPD. A linear regression model with oviposition rate as the dependent variable and trichome density and LMA as independent variables showed that both variables influenced the oviposition rate approximately equally. We conclude that oviposition was accelerated under low humidity (high VPD) conditions indirectly probably through an increase in the trichome density of host leaves.

Validation of a handheld near-infrared spectrophotometer for measurement of chemical intramuscular fat in Australian lamb.

The objective of the study was to independently validate a calibrated commercial handheld near infrared (NIR) spectroscopic device and test its repeatability over time using phenotypically diverse populations of Australian lamb. Validation testing in eight separate data sub-groups (n = 1591 carcasses overall) demonstrated that the NIR device had moderate precision (R2 = 0.4-0.64, RMSEP = 0.70-1.22%) but fluctuated in accuracy between experimental site demonstrated by variable slopes (0.50-0.94) and biases (-0.86-0.02). The repeatability experiment (n = 10 carcasses) showed that time to scan post quartering affected NIR measurement from 0 to 24 h (P < 0.001). On average, NIR IMF% was 0.97% lower (P < 0.001) at 24 h (4.01% ± 0.166), compared to 0 h. There was no difference (P > 0.05) between Time 0 and 1 h or Time 0 and 4 h or between replicate scans within each time point. This study demonstrated the SOMA NIR device could predict lamb chemical IMF% with moderate precision and accuracy, however additional work is required to understand how loin preparation, blooming and surface hydration affect NIR measurement.

Selection of microalgae suitable for culturing with digestate from methane fermentation.

The effects ofdigestate on the growth rates of Euglena gracilis, Chlorella vulgaris, and Dunaliella tertiolecta were investigated to select suitable microalgae for culturing with digestate from methane fermentation. Microalgae were cultured in an aqueous solution containing digestate at concentrations of 5%, 10%, 13%, 20%, 40%, 50%, and 100%, and Cramer-Myers (CM) solution as a control, at photosynthetic photon flux densities (PPFDs) of 75-150 micromol m(-2) s(-1) with continuous illumination at 30 degrees C. The number of cells was monitored daily, and specific growth rates (mu) were calculated as cellular multiplication rates. The maximum mu values of these species were greater in appropriate concentrations of digestate than in CM medium. The maximum mu values were 0.047 h(-1) in 10% digestate for E. gracilis, 0.065 h(-1) in 20% digestate for C. vulgaris, and 0.052 h(-1) in 50% digestate for D. tertiolecta at a PPFD of 150 micromol m(-2) s(-1). The mu of D. tertiolecta were 2.5 and 1.1 times higher than those of E. gracilis and C. vulgaris, respectively, in 50% digestate. These results demonstrated that these species could be cultured at high growth rates with diluted methane fermentation sludge and that, among these species, Dunaliella sp. was suitable for culturing at higher concentration of digestate under relatively low-level light conditions.

Relationship between body mass index of offspring and maternal smoking during pregnancy.

OBJECTIVE: To examine the relationship between maternal smoking during pregnancy and the body composition of offspring. SUBJECTS: Grade 4 elementary school children (n=1366; boys/girls, 724/642; 9-10 years old) were enrolled in this study. All parents answered a lifestyle questionnaire, and children underwent passive smoking tests. Urinary cotinine measurement and lifestyle screening test parameters (that is, body weight, body length, body mass index (BMI), obesity index (OI), blood tests for liver function and lipid profile and questions regarding maternal smoking and lifestyle) were evaluated in terms of their relationship with maternal smoking. In addition, urinary 8-hydroxydeoxyguanosine (8-OHdG) concentration was measured in 80 randomly selected children to assess its relationship with oxidative stress. RESULTS: Both BMI and OI were significantly higher in children whose mothers smoked during pregnancy than in those whose mothers never smoked (BMI: 17.2±2.7 vs 16.9±2.5 kg m(-2), P=0.016; OI: 2.7±14.3% vs 0.4±14.0%, P=0.003). The degree of elevation was positively correlated with the duration of maternal smoking. The increases in BMI and OI resulted from increased body weight and reduced height. The confounding factors-'breakfast with family', 'watching television at dinner', 'eating and drinking before sleep', 'watching television for >2 h', 'sleep duration <8 h' and 'playing sports'-were statistically significant. BMI and OI were significantly high in children whose mothers smoked during pregnancy in these six confounders. On the other hand, urinary 8-OHdG concentration was negatively correlated with BMI in children who had >1.3 ng ml(-1) urinary cotinine, suggesting that it may be related to basal metabolism due to oxidative stress. CONCLUSION: Maternal smoking is a risk factor for higher BMI and OI in 9- to 10-year-old children whose mothers smoke during pregnancy and may be independent of other confounding factors.

Erythroleukemia induction by Friend leukemia virus. A host gene locus controlling early anemia or polycythemia and the rate of proliferation of late erythroid cells.

This report confirms that the Fv-5 locus controls the types of erythropoiesis induced by Friend erythroleukemia virus (FLV) (21) and extends the study to investigate the mode of action of this locus. With the use of FLV obtained by a variety of procedures, we showed that the polycythemia spleen focus-forming component (SFFVp) was responsible for the contrasting changes of hematocrits observed in FV-Pp (polycythemia strain)-infected DBA/2 (Fv-5pp) or CBA (Fv-5aa) mice. These changes in hematocrits were found to be a direct result of the rise in circulating reticulocytes and erythrocytes in DBA/2 mice and a corresponding drop of these erythroid cells in CBA mice 2 wk after infection. Examination of the FV-P-induced cellular changes indicated that dramatic increase in erythropoietin (epo)-independent erythroid precursor (CFU-E*) cells was detected in the spleens and marrow of both strains of mice. The epo responsiveness of the CFU-E in the uninfected and FV-P-infected CBA and DBA/2 mice was also very similar. Similar to FLV-infected DBA/2 mice, the FV-P-infected CBA mice also developed tumorogenic cells (CFU-FV) relatively early after infection (4-6 wk). Study of the physiological and pathological changes in the marrows and spleens of these infected mice indicated that significant differences were found in the spleens of the two strains of mice. The percent of reticulocytes in the spleen cells of CBA mice remained between 10 and 20%, and level of the DBA/2 mice increased to approximately 50%. This higher rate of erythropoiesis was also reflected in the significantly higher rate of uptake of 59Fe in the spleens of the DBA/2 mice. These results suggest that the Fv-5 locus might control the hematocrit levels of these mice by regulating the rates of erythropoiesis in the spleen levels of these mice, probably by affecting the rate of proliferation of an erythroid cell or cells. The erythroid cell(s) affected is likely to be more mature than the erythroid progenitor, CFU-E, as the levels of CFU-E in these two strains of mice were similar. The hypothesis that Fv-5 may control the rates of proliferation of a late erythroid (cell(s) is also supported by the significantly higher spleen weights found in the infected DBA/2 (approximately 2.5 g/spleen) mice than in the CBA (approximately 1 g/spleen) strain.

Ecological divergence and habitat isolation between two migratory forms of Japanese threespine stickleback (Gasterosteus aculeatus).

When two closely related species migrate to divergent spawning sites, divergent use of spawning habitats can directly reduce heterospecific mating. Furthermore, adaptations to divergent spawning habitats can promote speciation as a by-product of ecological divergence. Here, we investigated habitat isolation and ecological divergence between two anadromous forms of threespine stickleback (Gasterosteus aculeatus), the Japan Sea and Pacific Ocean forms. In several coastal regions of eastern Hokkaido, Japan, these forms migrate to the same watershed to spawn. Our field surveys in a single watershed revealed that segregation of distinct spawning sites between the two forms was maintained within the watershed across multiple years. These spawning sites diverged in salinity and predator composition. Morphological and physiological divergence between the forms also occurs in the direction predicted by ecological differences between the spawning sites. Our data indicate that migration into divergent spawning habitats can be an important mechanism contributing to speciation and phenotypic divergence in anadromous fishes.

Effects of morphological characteristics of Cucumis sativus seedlings grown at different vapor pressure deficits on initial colonization of Bemisia tabaci (Hemiptera: Aleyrodidae).

We investigated the effects of morphological characteristics of cucumber, Cucumis sativus L., seedlings grown at different vapor pressure deficits (VPDs) on initial colonization of whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) biotype B. The seedlings were grown at a VPD of 3.8 or 0.4 kPa for 9 d at 30 degrees C under a photoperiod of 12:12 (L:D) h. Pairs of seedlings, one grown at high VPD and the other at low VPD, were then installed in cages with a VPD of 1.3 kPa, and adults of B. tabaci between 150 and 200 were released. Leaves of high-VPD seedlings had fewer B. tabaci than those of low-VPD seedlings 24 h after release. They also had higher relative chlorophyll content, were thicker and tougher, and had abaxial trichomes with higher density. Water stress caused by the high VPD probably decreased the initial colonization of B. tabaci through changes in these leaf morphological characteristics.

Efficient recycling of nutrients in modern and past hypersaline environments.

The biogeochemistry of hypersaline environments is strongly influenced by changes in biological processes and physicochemical parameters. Although massive evaporation events have occurred repeatedly throughout Earth history, their biogeochemical cycles and global impact remain poorly understood. Here, we provide the first nitrogen isotopic data for nutrients and chloropigments from modern shallow hypersaline environments (solar salterns, Trapani, Italy) and apply the obtained insights to δ15N signatures of the Messinian salinity crisis (MSC) in the late Miocene. Concentrations and δ15N of chlorophyll a, bacteriochlorophyll a, nitrate, and ammonium in benthic microbial mats indicate that inhibition of nitrification suppresses denitrification and anammox, resulting in efficient ammonium recycling within the mats and high primary productivity. We also suggest that the release of 15N-depleted NH3(gas) with increasing salinity enriches ammonium 15N in surface brine (≈34.0‰). Such elevated δ15N is also recorded in geoporphyrins isolated from sediments of the MSC peak (≈20‰), reflecting ammonium supply sufficient for sustaining phototrophic primary production. We propose that efficient nutrient supply combined with frequent bottom-water anoxia and capping of organic-rich sediments by evaporites of the Mediterranean MSC could have contributed to atmospheric CO2 reduction during the late Miocene.

Efficacy of perfusion cooling of the epidural space and cerebrospinal fluid drainage during repair of extent I and II thoracoabdominal aneurysm.

AIM: The aim of this study was to evaluate spinal cord injury and mortality resulting from repair of extent I and II thoracoabdominal aneurysm. The authors compared patients operated under mild hypothermia with or without epidural perfusion cooling (EPC) and cerebrospinal fluid drainage (CSFD). METHODS: From 1988 to 2007, 116 patients underwent replacement of the thoracoabdominal aorta; the procedure was performed in 38 patients with the aid of mild hypothermia alone (group A), and in 78 patients with the aid of EPC, mild hypothermia and CSFD (group B). Two catheters for epidural perfusion cooling were inserted in group B, in which one catheter was inserted into the epidural space to infuse chilled saline, and the other was inserted into the subdural space to drain the cerebrospinal fluid and to measure temperature and pressure. There were no significant differences in mean age, etiology of aortic disease, and aneurysm extent between the two groups. RESULTS: There were no significant differences in cardiopulmonary bypass time, the lowest nasopharyngeal temperature and operation time between the two study groups. The incidence of spinal cord injury in group A (16.2%) was significantly higher than in group B (3.8%, P=0.03). Hospital mortality in groups A and B was 10.5% and 2.6%, respectively (P=0.08). There was no significant difference in postoperative complications between the two study groups. CONCLUSION: The combination of EPC and CSFD was effective in lowering the incidence of postoperative spinal cord injury in the repair of extent I and II thoracoabdominal aortic aneurysm.

Interleukin 4 suppresses the spontaneous growth of chronic myelomonocytic leukemia cells.

We studied the effects of IL-4 on the spontaneous proliferation of chronic myelomonocytic leukemia (CMMoL) cells in vitro. IL-4 (100 U/ml) suppressed the spontaneous DNA synthesis by approximately 50% in 5 of 8 cases examined. IL-4 (100 U/ml) also inhibited the spontaneous colony formation by CMMoL cells in a methylcellulose culture by 50-97% in all of the 10 cases in which spontaneous colonies were formed. This IL-4-mediated suppression of the growth of CMMoL cells was completely abolished by the addition of anti-IL-4 neutralizing antibodies. The spontaneous CMMoL colonies were substantially suppressed by the addition of either anti-IL-6 or anti-granulocyte/macrophage colony-stimulating factor (GM-CSF) antibodies to the colony assay system: the addition of both anti-IL-6 and anti-GM-CSF antibodies resulted in greater than 80% inhibition of the colony formation by CMMoL cells. On the other hand, none of anti-IL-1-beta, anti-granulocyte-CSF, anti-macrophage-CSF, or anti-tumor necrosis factor-alpha antibodies affected the CMMoL colony formation. In the supernatants from 24-h cultures of CMMoL cells, high levels of IL-6 and GM-CSF were demonstrated in 9 of 9 and 2 of 9 cases examined, respectively. IL-4 (100 U/ml) almost completely inhibited the secretion of IL-6 and GM-CSF by CMMoL cells. These observations suggest that IL-4 suppresses the spontaneous proliferation of CMMoL cells by inhibiting their production of IL-6 and/or GM-CSF, both of which could act in vitro as an autocrine growth factor for CMMoL cells.

Deficiency of coagulation factor XIII A subunit caused by the dinucleotide deletion at the 5' end of exon III.

A congenital deficiency of the coagulation Factor XIII A subunit (F XIII A) is a rare autosomal recessive disorder that is characterized by a life-long bleeding tendency complicated by a difficulty in healing. Thus far, no molecular genetic analysis of this disorder has been reported. In this study, we demonstrate the molecular abnormalities in a family with this disorder. We performed Northern blot analysis of peripheral blood monocytes obtained from the propositus and found a 4-kb single band of F XIII A mRNA whose size was identical with that of normal subjects. Exons II-XV, which encode all the amino acids, were individually amplified by a polymerase chain reaction (PCR). All PCR products from the propositus had lengths indistinguishable from those of the wild type on agarose gel, suggesting that this defect results from either a point mutation or a short deletion/insertion. The sequencing of F XIII A cDNA from the propositus revealed a deletion of the dinucleotide AG within the AGAG repeat at the position of 210 to 213. Concerning the genomic sequence, a deletion of dinucleotide AG was also demonstrated in the intron B-exon III boundary. This deletion appeared to cause a frameshift mutation making a new stop codon shortly thereafter, and leading to a deficiency of plasma F XIII A. The heterozygosity of the F XIII A deficiency in the patient's offspring was documented by the nucleotide sequences of their exon III.

Morphological, immunological, and biochemical analyses of chicken spleen cells transformed in vitro by reticuloendotheliosis virus strain T.

Reticuloendotheliosis virus strain T (REV-T) is a highly oncogenic avian retrovirus which causes a rapid neoplastic disease of the lymphoreticular system. We derived six cell lines (1-3, 1-5, 2-10, 2-14, 2-16, and 2-20) from chicken spleen cells infected with REV-T. These cells can produce both the REV-T and its associated reticuloendotheliosis helper virus, REV-A. Histochemical analyses of these cells indicate that, while they are not stained by benzidine, peroxidase, beta-glucuronidase or acid alpha-naphthyl acetate esterase, they contain a high proportion (95%) of cells positive for acid phosphatase. Light and electron microscopic studies of these cells also revealed morphologies of lymphoblasts or activated lymphocytes with irregular nuclei and dispersed chromatin. Immunochemical analyses indicate that essentially all (90 to 100%) of the cells contain the surface marker Ia, but no cytoplasmic immunoglobulin M and immunoglobulin G could be detected by immunofluorescence staining. Results also show that some of these cell lines contain a low level of terminal transferase (0.02 to 0.17 unit/10(9) cells), and a proportion (3 to 35%) of these cells can be stained by an antiserum directed against chicken bursa cells. These results are consistent with the conclusion that the cells transformed by the highly oncogenic REV-T are lymphoid in nature. In addition, at least some of these cell clones may contain features characteristic of activated B-lymphocytes. Analysis of these cell clones indicates that some cell lines contain an adherent and nonadherent population with some differences in morphologies. In addition, electron microscopic examination revealed that, while the non-adherent cells are actively producing type C viruses, type C viruses are either absent or very rare in the adherent cell populations. These results support the conclusion that some of these cell lines are heterogeneous and contain subpopulations of cells with differences in their ability to produce viruses.

Effect of human recombinant granulocyte/macrophage colony-stimulating factor and native granulocyte colony-stimulating factor on clonogenic leukemic blast cells.

The effects of human recombinant granulocyte/macrophage colony-stimulating factor (GM-CSF) and human native purified granulocyte colony-stimulating factor (G-CSF) on the growth of clonogenic leukemic blast cells from eight Japanese patients with acute myeloblastic leukemia were studied, using an in vitro leukemic blast colony assay. The results showed that GM-CSF stimulated leukemic blast colony formation in all cases examined, whereas G-CSF stimulated colony formation in four of the eight cases. The maximum stimulating activity of GM-CSF on the growth of clonogenic leukemic blast cells was higher than that of G-CSF in the majority of cases, while sometimes GM-CSF and G-CSF worked synergistically. Thus, the clonogenic leukemic blast cell populations seemed to be heterogeneous with respect to their in vitro response to growth regulators.

Deletion of the carboxyl-terminal exons of K-sam/FGFR2 by short homology-mediated recombination, generating preferential expression of specific messenger RNAs.

The K-sam gene was first identified as an amplified gene from human gastric cancer cell line KATOIII, and its product is identical to fibroblast growth factor receptor 2. The K-sam gene is located on human chromosome 10q26 and is preferentially amplified in the poorly differentiated types, especially in the scirrhous type, of gastric cancers. During the course of studies on the structural characterization of the amplification units, we found that the carboxyl-terminal exons of K-sam were deleted in three of four of the scirrhous type of gastric cancer cell lines. These deletions generate preferential expression of mRNAs encoding K-sam proteins lacking the carboxyl-terminal region containing the tyrosine residues at positions 780, 784, and 813. The carboxyl-terminal region has been reported to have a sequence required for the inhibition of NIH3T3 transformation, indicating that cells with amplification of the truncated K-sam gene have a growth advantage during the carcinogenic process for the scirrhous type of gastric cancers. This is the first report showing the deletion of the carboxyl-terminal exons of the receptor-type of the protein tyrosine kinase gene. Sequence analysis of the DNA sequences surrounding the deletion junctions shows the presence of unique sequences and indicates the involvement of short homology-mediated recombination in the generation of these deletions.