Radiofrequency identification tag system improves the efficiency of closed vitrification for cryopreservation and thawing of bovine ovarian tissues.

PURPOSE: A radiofrequency identification (RFID) tag system was designed to streamline cryopreservation and thawing procedures. This study evaluated the usefulness of the RFID tag system for improving the efficiency of cryopreserving/thawing bovine ovarian tissue by the closed vitrification protocol. METHODS: Six participants carried out closed vitrification and thawing of bovine ovarian tissues procedures using either the conventional or the new RFID tag method, and the time required to perform each step of the respective methods was measured. After normality of data was confirmed by the Shapiro-Wilk test, the significance of differences was assessed by the unpaired t test. RESULTS: When closed vitrification was performed, the time required for each step showed a significant difference between the two methods (t(4) = 2.938, p = 0.042, d = 2.40), and the total cryopreservation time was 11 min shorter using the RFID tag system. When thawing was performed, the time required for each step also showed a significant difference between the two methods (t(4) = 2.797, p = 0.049, d = 2.28), and the total thawing time was 2 min shorter using the RFID tag system. CONCLUSION: The RFID tag system tested in this study seems to be suitable for managing biological samples stored in liquid nitrogen. Adoption of an RFID tag system by fertility centers may not only improve the efficiency of cryopreserving/thawing reproductive tissues but could also reduce human error.

BK Viremia as a Predictor of Hemorrhagic Cystitis in Adults During the First 100 Days After Allogeneic Hematopoietic Stem Cell Transplantation.

In a retrospective case-control study, we aimed to assess the utility of plasma BK viral load value to predict hemorrhagic cystitis (HC) symptoms after allogeneic hematopoietic stem cell transplantation (alloHSCT). During first 100 post-transplantation days of all adult AlloHSCT recipients at the University of Nebraska Medical Center from October 1, 2011, to June 30, 2014, 8 unexcluded cases of HC were identified and matched with 88 unexcluded unaffected control cases. Viral loads were determined for archived DNA extracted from plasma collected within 3 weeks before transplantation until ∼100 days after transplantation. Clinical factors, time of onset of BK viremia, and BK viral load were compared between case and control subjects to identify risks for HC. Symptomatic HC occurred in 8/96 (8.3%) of patients at a median of 34 days after transplantation. BK viremia either before or during symptoms was detected in all 8 (100%) HC patients and in 20/88 (22.7%) of control subjects. BK viremia was detected at a median of 8 days before HC clinical symptoms. The log of first positive viral load was not a statistically significant predictor (P = .17) of symptomatic BK. Median BK viral load peak was significantly higher for 8 patients with HC versus 20 viremic patients without HC (6.66 vs 5.06; P < .052). Further study is required to evaluate the predictive value of the BK viral load for HC.

Magnetic resonance cisternography for preoperative evaluation of arachnoid cysts.

INTRODUCTION: With a high likelihood of clinical improvement and low rates of complications, minimally invasive neuroendoscopic surgery is becoming the treatment of choice for symptomatic or growing arachnoid cysts. In neuroendoscopic surgery, visualization of anatomical landmarks is essential in achieving successful fenestration without complications. Because of the restricted visual field in neuroendoscopic surgery, preoperative anatomical assessment is very helpful. Magnetic resonance cisternography (MRC) with high spatial resolution and contrast, using for example 3-D Fourier transformation constructive interference in steady state (CISS) or fast imaging employing steady-state acquisition (FIESTA) sequences, is able to detect the arachnoid cyst wall and neighboring anatomical structures as the anatomical landmarks. We retrospectively reviewed T2-weighted (T2-W) fast spin-echo images, and the MRC and intraoperative findings. METHODS: Axial and coronal T2-W images (6 and 3 mm thickness, respectively) and axial and coronal 0.8 mm thick MRC images with CISS or FIESTA were obtained from four patients with arachnoid cysts treated by neuroendoscopic surgery. Intraoperative findings were reviewed on videotape recorded during the procedures. RESULTS: At the brain surface, the arachnoid cyst wall could be detected clearly in any of the four patients on MRC images, and was only partly seen in the fourth patient T2-W images. Adjacent important anatomical structures including vessels and cranial nerves, and an enough space for cystocisternostomy were identified on MRC images, and the findings were consistent with the findings during neuroendoscopic surgery. CONCLUSION: Preoperative identification of the arachnoid cyst wall and surrounding anatomical structures by MRC may help avoid complications and allow safer neuroendoscopic surgery.

cDNA structure of an insulin-related peptide in the Pacific oyster and seasonal changes in the gene expression.

Insulin-related peptide cDNA was characterized in the Pacific oyster Crassostrea gigas. It was determined that three transcripts with differing lengths of 3'-untranslated region (3'-UTR) were expressed in the visceral ganglia. The insulin-related peptide cDNA contained a number of AUUUA motifs that were typical of adenylate/uridylate-rich elements in the 3'-UTR. The deduced preprohormone was a polypeptide of 161 residues and showed a conformation typical of preprohormones of the insulin superfamily, which included conserved amino acids necessary to adopt the globular insulin structure. The expression of the three different transcripts was variable throughout the year, with the highest expression observed in March and lower expression in November and July. The expression of the shortest mRNA in March was about tenfold higher than in July, while the expression of the longest transcript varied approximately twofold during the year. The accumulation of glycogen in the soft body rapidly increased in October and November, and robust body growth and gametogenetic development occurred in March to May. The period of the highest expression of the oyster insulin-related peptide gene corresponded to the onset of body growth and gametogenetic development, but did not overlap with the period of glycogen accumulation. This is the first report that fully details the structure and expression of the insulin-related peptide gene in bivalves.

Nature of the well screened state in hard X-ray Mn 2p core-level photoemission measurements of La1-xSrxMnO3 films.

Using hard x-ray (HX; hnu=5.95 keV) synchrotron photoemission spectroscopy (PES), we study the intrinsic electronic structure of La(1-x)Sr(x)MnO(3) (LSMO) thin films. Comparison of Mn 2p core-levels with soft x-ray (SX; hnu approximately 1000 eV) PES shows a clear additional well-screened feature only in HX PES. Takeoff-angle dependent data indicate its bulk (> or =20 A) character. The doping and temperature dependence track the ferromagnetism and metallicity of the LSMO series. Cluster model calculations including charge transfer from doping-induced states show good agreement, confirming this picture of bulk properties reflected in Mn 2p core-levels using HX PES.

Zernike-type X-ray imaging microscopy at 25 keV with Fresnel zone plate optics.

A Zernike-type imaging microscope using a sputtered-sliced Fresnel zone plate (SS-FZP) has been developed and tested at an X-ray energy of 25 keV. The SS-FZP was used as an objective. A copper (Cu) phase plate was placed at the back focal plane of the SS-FZP in order to produce phase contrast. The performance of the Zernike-type imaging microscope was tested with a gold (Au) mesh and a resolution test pattern at undulator beamline 47 of SPring-8. The Au mesh and the resolution test pattern could be imaged in transmission with a magnification of x10.2. Owing to the Cu phase plate, different image contrast was observed compared with the bright-field image contrast. Tantalum microstructures down to 0.5 microm line-and-space have been observed on spatial resolution test patterns.

Development of a multilayer Fresnel zone plate for high-energy synchrotron radiation X-rays by DC sputtering deposition.

Hard X-ray microscopy with high spatial resolution (or=20 keV) because a large thickness (aspect ratio) can be available. Various types of multilayer FZPs have been fabricated by DC sputtering deposition. Their focusing characteristics have been evaluated at the high-brilliance undulator beamline BL47XU of SPring-8. An optical system using a Cu/Al multilayer FZP (with an outermost zone width of 0.25 microm) as the focusing optics fabricated by the optimum deposition condition with precise film (zone) thickness control has attained an almost diffraction-limited microbeam of 0.3-0.35 microm at 8.9 keV. A line-and-space resolution test pattern has been observed: fine structures up to 0.2 microm were clearly observed in the measured image. This FZP has been working since 1995, keeping good focusing characteristics. It can be said from these results that a spatial resolution better than 0.1 microm in the high-energy X-ray region is in prospect by the development of a multilayer FZP with a narrower outermost zone width in the near future.

Hard X-ray microbeam experiments with a sputtered-sliced Fresnel zone plate and its applications.

Hard X-ray microbeam experiments with sputtered-sliced Fresnel zone plates have been performed. Zone plates with an outermost zone width of 0.25 microm (#FZP1) and 0.1 microm (#FZP2) were fabricated and evaluated. In a scanning X-ray microscopy experiment, a line-and-space pattern with structure as fine as 0.1 microm was resolved using #FZP2 at an X-ray wavelength of 1 A. As an application of the microbeam technique, a two-dimensional distribution of constituent elements in forensic samples has been obtained (e.g. section view of human and elephant hairs) using fluorescent scanning microscopy.

Monolayer formation and DNA synthesis of the outer epithelial cells from pearl oyster mantle in coculture with amebocytes.

In vitro experiments were conducted to clarify the involvement of the epithelium-amebocyte interaction in epithelial regeneration of bivalves. The outer epithelia of the pallial mantle of the pearl oyster. Pinctada fucata martensii, were separated in cell sheets from the inner connective tissue layers by digestion with Dispase. Clumps of the separated mantle epithelia were inoculated onto the amebocyte layers prepared on the bottom of culture dishes and maintained at 20 degrees C in 5% CO2:95% air for 1 wk. Balanced salt solution with 0.03% (wt/vol) glucose was used as a culture medium. The epithelial cells adhered to the amebocyte layers within 24 h, changed their shape from cuboidal to squamous, and migrated and formed monolayer sheets within 3 d. Electron microscopy confirmed maintenance of epithelial polarity and cell to cell junction in the sheets; 6 d after the inoculation, 5-bromo-2'-deoxyuridine was added to the culture at 30 microns. After labeling for 24 h, the cultures were fixed and stained with anti 5-bromo-2'-deoxyuridine antibody. Cells with immunoreactive nuclei were clearly observed in the epithelial cell sheets, indicating active DNA synthesis in the epithelial sheets. Thus, cocultured with amebocytes, the outer epithelial cells from pallial mantle tissue formed a monolayer sheet and started DNA synthesis. The morphological features of the mantle outer epithelial cells are analogous to those described for the in vivo cutaneous wound healing process, suggesting that the epithelium-amebocyte interaction is important in the regeneration of epithelium in bivalves.

Reduction of adhesions with fibrin glue after laparoscopic excision of large ovarian endometriomas.

STUDY OBJECTIVE: To evaluate the effect of fibrin glue on the formation of adhesions after laparoscopic excision of endometriomas. DESIGN: Prospective case series. SETTING: Department of Obstetrics and Gynecology at a university-affiliated hospital. PATIENTS: Thirty women with ovarian cysts. INTERVENTIONS: In 10 women (16 cysts) we performed laparoscopic resection of endometriomas leaving the ovarian capsule open. Twenty patients (25 cysts) underwent laparoscopic removal of endometriomas and fibrin glue approximation of the ovarian capsule, as well as coating of serosal defects with the glue. At second-look laparoscopy 4 to 6 months after the initial surgery, postoperative adhesion formation was assessed according to the revised American Fertility Society classification. MEASUREMENTS AND MAIN RESULTS: The mean (+/- SEM) adnexal adhesion score in patients without fibrin glue was 8.6 +/- 2.2 at initial surgery and increased slightly to 12.6 +/- 2.6 at second-look laparoscopy. In patients treated with fibrin glue, the score decreased significantly from 10.9 +/- 1.4 to 7.8 +/- 1.3 (p <0.02). The decrease was most marked in women with endometriomas 5 cm or more in diameter and for those with a preoperative adhesion score of 8 or higher. CONCLUSION: Fibrin glue reduced postoperative adhesions after laparoscopic resection of endometriomas.

Effects of transgenic mixed-haplotype MHC class II molecules A alpha d A beta z on autoimmune disease in New Zealand mice.

The MHC haplotype heterozygosity (H-2d/H-2z) acts as one major predisposing genetic element for autoimmune disease resembling systemic lupus erythematosus (SLE) in the F1 hybrid of NZB (H-2d) and NZW (H-2z) mice. To determine a possible role of mixed-haplotype A molecules, we introduced a transgene A beta z into H-2d/H-2d homozygous (NZB x NZW.H-2d)F1 mice, in which, compared with the original H-2d/H-2z heterozygotes, the incidence and titer of IgG anti-DNA antibodies, serum levels of nephritogenic retroviral gp70/anti-gp70 immune complexes (ICs) and the associated incidence of IC-type lupus nephritis were reduced. Evidence for the formation of mixed-haplotype A alpha d A beta z molecules in the transgenic mice was obtained by A beta z molecule expression on the cell surface of splenic B cells and macrophages, thymic epithelial cells and mature B cells in the bone marrow. A alpha d A beta z-restricted T cell clones showed good proliferative responses to spleen cells from the transgenic mice, to an extent much larger than seen in cells from the original (NZB x NZW)F1 mice, suggesting that the expression of functional A alpha d A beta z molecules on cells in transgenic mice is considerably high. Compared with findings in transgene-negative littermates and the original (NZB x NZW)F1 mice, the A beta z transgene to a greater extent promoted serum levels of IgM anti-double-stranded (ds) DNA antibodies and IgM anti-gp70/gp70 ICs in the transgenic mice. None the less, the production of IgG anti-dsDNA antibodies and IgG anti-gp70/gp70 ICs as well as the development of renal disease was markedly suppressed. Possible mechanisms of such effects of the transgene are discussed.

Exogenous Thy-1.1 expression as a marker for thymocyte maturation in transgenic mice.

We established a line of transgenic mice carrying the exogenous mouse Thy-1.1 gene (8.2 Kb Eco RI genomic DNA fragment). In these mice, Thy-1.1 was expressed on thymocytes but not on peripheral T cells, presumably due to the lack of cis-acting element(s) on the microinjected genomic DNA. Even in the thymus, however, while most of the CD3/TCR- thymocytes were positive for the transgenic Thy-1.1 gene expression, the CD4+ or CD8+ single positive (SP) thymocytes were composed of two groups, one Thy-1.1+ and one Thy-1.1-, suggesting that the former belongs to cells at premature stages of terminal T cell differentiation. There was no difference in the amount of CD3/TCR complexes expressed on two such SP thymocyte subsets. In the double negative (DN) thymocytes, all the CD3/TCR+ cells were Thy-1.1-. These results suggest that the maturational process of CD3+ DN thymocytes differs from that of SP thymocytes. The unique distribution of Thy-1.1+ population among CD3+ thymocytes suggests that the transgenic Thy-1.1 gene expression can serve as a useful marker to examine terminal maturation processes of CD3+ thymocytes.