Germination of the fully myco-heterotrophic orchid Cyrtosia septentrionalis is characterized by low fungal specificity and does not require direct seed-mycobiont contact

We examined the level of specificity exhibited by seeds of Cyrtosia septentrionalis for germination-inducing fungi. Three wood-decomposing Armillaria species (A. gallica, A. mellea subsp. nipponica and A. tabescens) known to colonize adult plants and an unidentified species of Polyporales isolated from naturally growing protocorms were tested. Xylobolus annosus, a free-living decomposer of Russulales, was included as a control. Seed germination occurred in a sawdust-based medium in sealed and unsealed containers in the presence of all fungi, indicating low mycorrhizal specificity in germination. Moreover, germination occurred even in modified containers in which the fungus was physically isolated from the orchid seeds, indicating that direct seed-fungus contact is not required. Higher germination percentages were observed in sealed containers in which a modified atmosphere, consisting of a lower O₂ and a higher CO₂ concentration, had been established in the air above the inoculated medium as a result of the saprophytic activity of the fungus. In nature, atmospheric conditions more effective for seed germination might be established by the action of mycobionts in decomposing wood. Seeds germinated in the presence of the unknown Polyporales species underwent further growth and development, as compared with the other fungi tested, when covered with soil.     

MicroRNA profile predicts recurrence after resection in patients with hepatocellular carcinoma within the Milan Criteria

Objective

Hepatocellular carcinoma (HCC) is difficult to manage due to the high frequency of post-surgical recurrence. Early detection of the HCC recurrence after liver resection is important in making further therapeutic options, such as salvage liver transplantation. In this study, we utilized microRNA expression profiling to assess the risk of HCC recurrence after liver resection.

Methods

We examined microRNA expression profiling in paired tumor and non-tumor liver tissues from 73 HCC patients who satisfied the Milan Criteria. We constructed prediction models of recurrence-free survival using the Cox proportional hazard model and principal component analysis. The prediction efficiency was assessed by the leave-one-out cross-validation method, and the time-averaged area under the ROC curve (ta-AUROC).

Results

The univariate Cox analysis identified 13 and 56 recurrence-related microRNAs in the tumor and non-tumor tissues, such as miR-96. The number of recurrence-related microRNAs was significantly larger in the non-tumor-derived microRNAs (N-miRs) than in the tumor-derived microRNAs (T-miRs, P<0.0001). The best ta-AUROC using the whole dataset, T-miRs, N-miRs, and clinicopathological dataset were 0.8281, 0.7530, 0.7152, and 0.6835, respectively. The recurrence-free survival curve of the low-risk group stratified by the best model was significantly better than that of the high-risk group (Log-rank: P = 0.00029). The T-miRs tend to predict early recurrence better than late recurrence, whereas N-miRs tend to predict late recurrence better (P<0.0001). This finding supports the concept of early recurrence by the dissemination of primary tumor cells and multicentric late recurrence by the ‘field effect’.

Conclusion

microRNA profiling can predict HCC recurrence in Milan criteria cases.     

Histone H3 messenger RNA in situ hybridization for identifying proliferating cells in formalin-fixed rat gastric mucosa

To devise a more sensitive method for identifying proliferative cells in routinely formalin-fixed, paraffin-embedded tissues, we applied an in situ hybridization (ISH) technique for the detection of histone H3 mRNA in rat gastric mucosa and amplified the signal by a silver intensification method. ISH was performed using a Fluorescein-labelled, single-stranded DNA probe for the human histone H3 gene. To determine the optimal conditions for detecting H3 mRNA in rat gastric mucosa, we tested the effect of changing conditions, such as fixation time and digestion time, by a proteinase before hybridization. Next, the proliferation indices obtained using H3 ISH were compared with those obtained using bromodeoxyuridine (BrdU) immunohistochemistry. In normal rat gastric mucosa, H3 ISH- and BrdU-positive cells were confined to the neck region of both fundic and pyloric mucosa. The two labelling indices were almost the same. In all the serial sections studied, H3 ISH-positive cells were almost always BrdU-positive too. Taken together, these results indicate that the H3 ISH technique is useful for the evaluation of proliferative activity in gastric epithelial cells by virtue of its detection of S-phase cells This revised version was published online in November 2006 with corrections to the Cover Date.     

Burrowing criteria and burrowing mode adjustment in bivalves to varying geoenvironmental conditions in intertidal flats and beaches

The response of bivalves to their abiotic environment has been widely studied in relation to hydroenvironmental conditions, sediment types and sediment grain sizes. However, the possible role of varying geoenvironmental conditions in their habitats remains poorly understood. Here, we show that the hardness of the surficial intertidal sediments varies by a factor of 20-50 due to suction development and suction-induced void state changes in the essentially saturated states of intertidal flats and beaches. We investigated the response of two species of bivalves, Ruditapes philippinarum and Donax semigranosus, in the laboratory by simulating such prevailing geoenvironmental conditions in the field. The experimental results demonstrate that the bivalve responses depended strongly on the varying geoenvironmental conditions. Notably, both bivalves consistently shifted their burrowing modes, reducing the burrowing angle and burial depth, in response to increasing hardness, to compensate for the excessive energy required for burrowing, as explained by a proposed conceptual model. This burrowing mode adjustment was accompanied by two burrowing criteria below or above which the bivalves accomplished vertical burrowing or failed to burrow, respectively. The suitable and fatal conditions differed markedly with species and shell lengths. The acute sensitivities of the observed bivalve responses to geoenvironmental changes revealed two distinctive mechanisms accounting for the adult-juvenile spatial distributions of Ruditapes philippinarum and the behavioral adaptation to a rapidly changing geoenvironment of Donax semigranosus. The present results may provide a rational basis by which to understand the ensuing, and to predict future, bivalve responses to geoenvironmental changes in intertidal zones.     

Isolation of a new cellulase gene from a thermophilic anaerobe and its expression inEscherichia coli

Summary A new cellulase gene was cloned and expressed inEscherichia coli from a thermophilic anaerobe, strain NA10. A 7.4 kbEcoRI fragment of NA10 DNA encoded the cellulase which hydrolyzed carboxymethyl cellulose, lichenan, andp-nitrophenyl--d-cellobioside, but could not digest laminarin andp-nitrophenyl--d-glucoside. The cloned enzyme could digest cellooligosaccharides and release cellobiose as a main product from cellotetraose but could not digest cellobiose. It was distinct from the endoglucanase which was cloned by us previously from NA10 strain in terms ofp-nitrophenyl--d-cellobioside degradation activity and the location of restriction enzyme sites. The enzyme produced byE. coli transformant was extremely heat-stable and the optimum temperature for the enzymatic reaction was 80°C. Fifty three percent of the cloned enzyme was detected in the periplasm and the remaining activity existed in the cellular fraction in theE. coli transformant.     

Agarose–gelatin conjugate for adherent cell-enclosing capsules

Spherical capsules were prepared by extruding aqueous agarose–gelation conjugate solution into co-flowing liquid paraffin at 38°C and cooling the resultant emulsion. Capsule diameter was controlled between 40 and 250 μm by changing the velocity of the liquid paraffin. Adherent Crandall–Reese feline kidney cells enclosed in conjugate capsules of 141 ± 23 μm diam. had a higher degree of proliferation than those in unmodified agarose capsules. Mitochondrial activity, detected for cell-enclosing conjugate capsules normalized against unit volume of gel, was about double that of unmodified agarose capsules over 28 days. These results demonstrated the feasibility of agarose–gelatin conjugate as a material of cell-enclosing capsules.     

Host plant architecture affects the costs of dropping behaviour in <Emphasis Type="Italic">Phaedon brassicae</Emphasis> (Coleoptera: Chrysomelidae)

Phytophagous insects escape from predators by dropping. However, if they drop to the ground, they must then return to the host plants. Large oval leaves may serve as safety nets when insects drop from overlying leaves. To clarify the effects of leaf shape on the sites to which insects drop, we investigated the dropping behaviour of Phaedon brassicae (Coleoptera: Chrysomelidae) on three host plants (daikon, bok choy, and Chinese cabbage). Daikon plants have cleft leaves, whereas bok choy and Chinese cabbage plants have oval leaves. When poked with forceps, larvae dropped less frequently than adults. The proportions of individuals dropping to the ground also differed among host plants. Both larvae and adults on the ventral (abaxial) leaf surfaces of daikon frequently dropped to the ground via clefts in the underlying leaves. However, larvae and adults on the ventral leaf surfaces of bok choy and Chinese cabbage frequently dropped to underlying leaves. Most larvae and adults that dropped to the ground finally returned to host plants. However, the return times were longer for larvae than adults. Therefore, the cost of dropping from daikon leaves was higher than were the costs of dropping from leaves of other crop species.