In vitro maturation and ultrastructural observation of cryopreserved minke whale (Balaenoptera acutorostrata) follicular oocytes

Minke whale (Balaenoptera acutorostrata) follicular oocytes were cryopreserved by a slow-step freezing procedure using ethylene glycol. The morphologically viable proportion of postthawed minke whale follicular oocytes was 39.7%. The maturity of the animals (immature and mature whales) or the presence or absence of cumulus cells (CC) did not affect the proportion of morphologically viable oocytes. Postthawed oocytes were examined for nuclear status after in vitro maturation. The presence of CC (29.1%) significantly enhanced (P < 0.05) the proportion of oocytes at metaphase I/anaphase I/telophase I stages compared to results with the absence of CC (13.5%). A total of 4 of 194 postthawed oocytes matured to the second metaphase stage after culture for 5.5 days with or without CC. The cryopreserved immature oocytes obtained from immature and mature whales were processed to examine the ultrastructure by transmission electron microscopy. Varying ultrastructural damage to the cytoplasm was observed as a result of the cryopreservation procedures. These results show that 20-30% of cryopreserved minke whale follicular oocytes can resume meiosis in vitro, but damage induced by the freezing and thawing procedures was observed.     

Molecular breeding of yeast with higher metal-adsorption capacity by expression of histidine-repeat insertion in the protein anchored to the cell wall

A fusion protein of hexa-histidine repeat (His) and glycosylphosphatidylinositol (GPI)-anchor region of Saccharomyces cerevisiae Cwp1 with Aspergillus oryzae Taka-amylase A (TAA) was expressed on the yeast cell surface. The expressed fusion protein (TAA-His-Cwp1) was localized on the cell wall and demonstrated amylolytic activity. In comparison with the TAA-Cwp1 expressing strain, these cells exhibited 1.6- to 2.8-fold higher adsorbing capacity for Cu(2+), Ni(2+), and Zn(2+).     

Cell Reproduction and Morphological Changes in Mycoplasma capricolum

The cell reproduction of Mycoplasma capricolum was studied. The velocity of DNA replication fork progression was about 6 kb/min, which is 10 times slower than that of Escherichia coli. The time required for one round of DNA replication accorded with the doubling time. The origin/terminus ratio was 2.0. M. capricolum cell morphology was classified into two types, rod and branched. In the ordinary-growth phase, the rod cells accounted for about 90% of the total population, with branched cells comprising the remaining 10%. The proportion of branched cells increased to 90% following inhibition of DNA replication by nucleoside starvation. An increase in the proportion of branched cells was induced by transfer of a temperature-sensitive mutant deficient in DNA replication to the restrictive temperature. The rod cells had a regular structure, a fixed cell length, and constrictions in the center. The DNA contents of individual rod cells were distributed with a standard deviation of 0.40 of average. The branched cells had irregular structures and a wide distribution of DNA contents. Counting of viable cells revealed that the cells ceased division upon cell type conversion; however, branched cells maintained a reproductive capacity. A model for the reproduction process is proposed.Mycoplasmas are parasitic bacteria that have extremely low G+C contents and small genomes (9). Their morphology is irregular because of the lack of a peptidoglycan layer.In Escherichia coli, initiation of chromosomal DNA replication occurs once during the cell’s replicative cycle, and the nucleoids partition before cell division (13). The chromosomal replication of E. coli initiates in a small region and proceeds in both directions. It is mainly controlled by the timing and frequency of initiation, while the velocity of replication is constant.In mycoplasmas, chromosome replication also starts at a fixed site, followed by bidirectional progression (19–21, 25, 40). As in many eubacteria (36), the dnaA gene is expressed and plays important roles in the initiation of replication (35). These observations suggest that the outline of chromosome replication of mycoplasmas is similar to that of E. coli. However, the process of mycoplasma cell reproduction has not been clarified. Moreover, the cell division cycle of E. coli cannot be simply applied to mycoplasmas because of their irregular cell morphology (4). A model has been suggested for the cell cycle of Mycoplasma mycoides (6, 30, 31), which is closely related to Mycoplasma capricolum (39). According to this model, an elementary rounded body grows into a filamentous form and then new elementary rounded bodies are developed within this filament and released, but this model has not been adequately substantiated.In this study, we analyzed the process of DNA replication, cell morphology, and viability under various conditions of M. capricolum and proposed a model of cellular reproduction for this bacterium.     

A novel screening method for microorganisms that efficiently remove heavy metals from aqueous solution

A novel bioassay system for estimating concentrations of several heavy metal ions was carried out with yeast mutants which are highly sensitive to heavy metal ions. The method does not need an atomic adsorption spectrometer or other special equipment. It is suitable for screening of microorganisms that efficiently remove heavy metal ions from aqueous solution.     

Flexible Ion‐Conducting Composite Membranes for Lithium Batteries

The use of metallic lithium anodes enables higher energy density and higher specific capacity Li‐based batteries. However, it is essential to suppress lithium dendrite growth during electrodeposition. Li‐ion‐conducting ceramics (LICC) can mechanically suppress dendritic growth but are too fragile and also have low Li‐ion conductivity. Here, a simple, versatile, and scalable procedure for fabricating flexible Li‐ion‐conducting composite membranes composed of a single layer of LICC particles firmly embedded in a polymer matrix with their top and bottom surfaces exposed to allow for ionic transport is described. The membranes are thin (<100 μm) and possess high Li‐ion conductance at thicknesses where LICC disks are mechanically unstable. It is demonstrated that these membranes suppress Li dendrite growth even when the shear modulus of the matrix is lower than that of lithium. It is anticipated that these membranes enable the use of metallic lithium anodes in conventional and solid‐state Li‐ion batteries as well as in future Li? S and Li? O₂ batteries.     

Unavoidable Human Errors of Tumor Size Measurement during Specimen Attachment after Endoscopic Resection: A Clinical Prospective Study

Objective

Objective evaluation of resected specimen and tumor size is critical because the tumor diameter after endoscopic submucosal dissection affects therapeutic strategies. In this study, we investigated whether the true tumor diameter of gastrointestinal cancer specimens measured by flexible endoscopy is subjective by testing whether the specimen is correctly attached to the specimen board after endoscopic submucosal dissection resection and whether the size differs depending on the endoscopist who attached the specimen.

Methods

Seventy-two patients diagnosed with early gastric cancer who satisfied the endoscopic submucosal dissection expanded-indication guideline were enrolled. Three endoscopists were randomly selected before every endoscopic submucosal dissection. Each endoscopist separately attached the same resected specimen, measured the maximum resection diameter and tumor size, and removed the lesion from the attachment board.

Results

The resected specimen diameters of the 3 endoscopists were 44.5±13.9 mm (95% Confidence Interval (CI): 23–67), 37.4±12.0 mm (95% CI: 18–60), and 41.1±13.3 mm (95% CI: 20–63) mm. Comparison among 3 groups (Kruskal Wallis H- test), there were significant differences (H = 6.397, P = 0.040), and recorded tumor sizes were 38.3±13.1 mm (95% CI: 16–67), 31.1±11.2 mm (95% CI: 12.5–53.3), and 34.8±12.8 (95% CI: 11.5–62.3) mm. Comparison among 3 groups, there were significant differences (H = 6.917, P = 0.031).

Conclusions

Human errors regarding the size of attached resected specimens are unavoidable, but it cannot be ignored because it affects the patient’s additional treatment and/or surgical intervention. We must develop a more precise methodology to obtain accurate tumor size.

Trial Registration

University hospital Medical Information Network UMIN No. 000012915     

Increase in Ultrasonic Intensity of Blood Speckle across Moderate Coronary Artery Stenosis Is an Independent Predictor of Functional Coronary Artery Stenosis Measured by Fractional Flow Reserve: Pilot Study

Background and Aims

The degree of coronary artery stenosis should be assessed both anatomically and functionally. We observed that the intensity of blood speckle (IBS) on intravascular ultrasound (IVUS) is low proximal to a coronary artery stenosis, and high distal to the stenosis. We defined step-up IBS as the distal minus the proximal IBS, and speculated that this new parameter could be used for the functional evaluation of stenosis on IVUS. The aims of this study were to assess the relationships between step-up IBS and factors that affect coronary blood flow, and between step-up IBS and fractional flow reserve (FFR).

Methods and Results

This study enrolled 36 consecutive patients with angina who had a single moderate stenosis in the left anterior descending artery. All patients were evaluated by integrated backscatter IVUS and intracoronary pressure measurements. FFR was calculated from measurements using a coronary pressure wire during hyperemia. Conventional gray-scale IVUS images were recorded, and integrated backscatter was measured in three cross-sectional slices proximal and distal to the stenosis. Step-up IBS was calculated as (mean distal integrated backscatter value) − (mean proximal integrated backscatter value). Stepwise multiple linear regression analysis showed that the heart rate (r = 0.45, P = 0.005), ejection fraction (r = −0.39, P = 0.01), and hemoglobin level (r = −0.32, P = 0.04) were independently correlated with step-up IBS, whereas proximal and distal IBS were not associated with these factors. There was a strong inverse correlation between step-up IBS and FFR (r = −0.84, P < 0.001), which remained significant on stepwise multiple linear regression analysis.

Conclusions

The newly defined parameter of step-up IBS is potentially useful for the functional assessment of coronary artery stenosis.