Prediction of the estrous cycle and optimal insemination time by monitoring vaginal electrical resistance (VER) in order to improve the reproductive efficiency of the Okinawan native Agu pig

The present study was conducted to determine whether vaginal electrical resistance (VER) can be exploited to improve the low reproductive efficiency of the rare Okinawan native Agu pig, in which estrous signs are difficult to ascertain by visual observation. The lowest VER (272.0+/-12.4 units, n=5) and the preovulatory LH surge were detected at 57.6+/-5.3 and 36.8+/-9.6h before the onset of estrus, respectively. The initiation of gradual increase in VER was found after 9.6+/-4.7h following the peak LH, and the higher levels of VER were plateaued during the luteal phase. These VER fluctuations were correlated with changes in plasma LH (P<0.05) and progesterone (P<0.001), but not estrogen. Moreover, the conception rate (41%, n=32) was dramatically improved by artificial insemination at 24 and 34 h after the beginning of the VER increase when compared with insemination at the conventional time (12 and 24h after detection of estrus, 20%, n=45), widely used in commercial pigs (P<0.05). These data suggest that VER fluctuation can be used to estimate the stage of the estrous cycle, and the scheduling artificial insemination according to increase in VER as an index for the preovulatory LH surge could improve Agu reproductive efficacy.     

A minimal base‐pairing region of a bacterial small RNA SgrS required for translational repression of ptsG mRNA

Escherichia coli SgrS is an Hfq‐binding small RNA that is induced under glucose‐phosphate stress to cause translational repression and RNase E‐dependent rapid degradation of ptsG mRNA encoding the major glucose transporter. A 31‐nt‐long stretch in the 3′ region of SgrS is partially complementary to the translation initiation region of ptsG mRNA. We showed previously that SgrS alone causes translational repression when pre‐annealed with ptsG mRNA by a high‐temperature treatment in vitro. Here, we studied translational repression of ptsG mRNA in vitro by synthetic RNA oligonucleotides (oligos) to define the SgrS region required for translational repression. We first demonstrate that a 31 nt RNA oligo corresponding to the base‐pairing region is sufficient for translational inhibition of ptsG mRNA. Then, we show that RNA oligo can be shortened to 14 nt without losing its effect. Evidence shows that the 14 nt base‐pairing region is sufficient to inhibit ptsG translation in the context of full‐length SgrS in vivo. We conclude that SgrS 168–181 is a minimal base‐pairing region for translational inhibition of ptsG mRNA. Interestingly, the 14 nt oligo efficiently inhibited ptsG translation without the high‐temperature pre‐treatment, suggesting that remodelling of structured SgrS is an important mechanism by which Hfq promotes the base pairing.     

Synthesis and optimization of hyaluronic acid–methotrexate conjugates to maximize benefit in the treatment of osteoarthritis

We previously reported that a conjugate of hyaluronic acid (HA) and methotrexate (MTX) could be a prototype for future osteoarthritis drugs having the efficacy of the two clinically validated agents but with a reduced risk of the systemic side effects of MTX by using HA as the drug delivery carrier. To identify a clinical candidate, we attempted optimization of a lead, conjugate 1. Initially, in fragmentation experiments with cathepsins, we optimized the peptide part of HA–MTX conjugates to be simpler and more susceptible to enzymatic cleavage. Then we optimized the peptide, the linker, the molecular weight, and the binding ratio of the MTX of the conjugates to inhibit proliferation of human fibroblast-like synoviocytes in vitro and knee swelling in rat antigen-induced monoarthritis in vivo. Consequently, we found conjugate 30 (DK226) to be a candidate drug for the treatment of osteoarthritis.     

Inhibition of hyaluronan synthesis in Streptococcus equi FM100 by 4-methylumbelliferone.

As observed previously in cultured human skin fibroblasts, a decrease of hyaluronan production was also observed in group C Streptococcus equi FM100 cells treated with 4-methylumbelliferone (MU), although there was no effect on their growth. In this study, the inhibition mechanism of hyaluronan synthesis by MU was examined using Streptococcus equi FM100, as a model. When MU was added to a reaction mixture containing the two sugar nucleotide donors and a membrane-rich fraction as an enzyme source in a cell-free hyaluronan synthesis experiment, there was no change in the production of hyaluronan. On the contrary, when MU was added to the culture medium of FM100 cells, hyaluronan production in the isolated membranes was decreased in a dose-dependent manner. However, when the effect of MU on the expression level of hyaluronan synthase was examined, MU did not decrease either the mRNA level of the has operon containing the hyaluronan synthase gene or the protein level of hyaluronan synthase. Solubilization of the enzyme from membranes of MU-treated cells and addition of the exogenous phospholipid, cardiolipin, rescued hyaluronan synthase activity. In the mass spectrometric analysis of the membrane phospholipids from FM100 cells treated with MU, changes were observed in the distribution of only cardiolipin species but not of the other major phospholipid, PtdGro. These results suggest that MU treatment may cause a decrease in hyaluronan synthase activity by altering the lipid environment of membranes, especially the distribution of different cardiolipin species, surrounding hyaluronan synthase.     

Expression of vinexin alpha in the dorsal half of the eye and in the cardiac outflow tract and atrioventricular canal

The Smads are intracellular signalling molecules that transduce signals from receptors for members of the TGF-beta superfamily to the nucleus. We have cloned the Xenopus orthologue of Smad3 (XSmad3). It is 94.6% identical to human Smad3 at the amino acid level. It is expressed as a maternal mRNA which disappears after stage 10.5, but reappears at the early tailbud stages. It is much less abundant than XSmad2 at the early developmental stages. From Stage 27 onwards XSmad3 is expressed with XSmad2 throughout the head region and in the somitic region. Strikingly however, XSmad3 alone is specifically expressed in the chordoneural hinge, the notochord and in the developing heart. Closer analysis reveals that XSmad3 is specifically expressed in the endocardium but not in the myocardium or pericardium. The chordoneural hinge staining persists at least until stage 40 whereas the staining in the endocardium peaks at approximately stage 32/33.     

Hunner-Type (Classic) Interstitial Cystitis: A Distinct Inflammatory Disorder Characterized by Pancystitis,with Frequent Expansion of Clonal B-Cells and Epithelial Denudation

Interstitial cystitis (IC) is a chronic bladder disease with urinary frequency, bladder discomfort or bladder pain of unknown etiology. Based on cystoscopic findings, patients with IC are classified as either Hunner-type/classic IC (HIC), presenting with a specific Hunner lesion, or non-Hunner-type IC (NHIC), presenting with no Hunner lesion, but post-hydrodistension mucosal bleeding. Inflammatory cell infiltration, composed predominantly of lymphocytes, plasma cells and epithelial denudation, has in the past been documented as a major pathological IC finding. However, the significance of the pathological evaluation of IC, especially with regard to the difference between HIC and NHIC, has been downplayed in recent years. In this study, we performed immunohistochemical quantification of infiltrating T-lymphocytes, B-lymphocytes and plasma cells, and measured the amount of residual epithelium in urinary bladder biopsy specimens taken from patients with HIC and NHIC, and those with no IC, using image analysis software. In addition, in situ hybridization of the light chains was performed to examine clonal B-cell expansion. Lymphoplasmacytic infiltration was significantly more severe in HIC specimens than in NHIC specimens (P <0.0001). Substantial lymphoplasmacytic inflammation (≥200 cells/mm²) was observed in 93% of HIC specimens, whereas only 8% of NHIC specimens were inflamed. Plasmacytic infiltration was more prominent in HIC specimens compared with NHIC and non-IC cystitis specimens (P <0.005). Furthermore, expansion of light-chain-restricted B-cells was observed in 31% of cases of HIC. The amount of residual epithelium was decreased in HIC specimens compared with NHIC specimens and non-IC cystitis specimens (P <0.0001). These results suggest that NHIC and HIC are distinct pathological entities, with the latter characterized by pancystitis, frequent clonal B-cell expansion and epithelial denudation. An abnormality in the B-cell population may be involved in the pathogenesis of HIC.     

Radical Prostatectomy versus External Beam Radiotherapy for cT1-4N0M0 Prostate Cancer: Comparison of Patient Outcomes Including Mortality

Background

Although radical prostatectomy (RP) and external beam radiotherapy (EBRT) have been considered as comparable treatments for localized prostate cancer (PC), it is controversial which treatment is better. The present study aimed to compare outcomes, including mortality, of RP and EBRT for localized PC.

Methods

We retrospectively analyzed 891 patients with cT1-4N0M0 PC who underwent either RP (n = 569) or EBRT (n = 322) with curative intent at our single institution between 2005 and 2012. Of the EBRT patients, 302 (93.8%) underwent intensity-modulated radiotherapy. Primary endpoints were overall survival (OS) and cancer-specific survival (CSS). Related to these, other-cause mortality (OCM) was also calculated. Biochemical recurrence-free survival was assessed as a secondary endpoint. Cox proportional hazards model was used for multivariate analysis.

Results

Median follow-up durations were 53 and 45 months, and median ages were 66 and 70 years (P <0.0001), in the RP and EBRT groups, respectively. As a whole, significantly better prognoses of the RP group than the EBRT group were observed for both OS and CSS, although OCM was significantly higher in the EBRT group. There was no death from PC in men with low and intermediate D’Amico risks, except one with intermediate-risk in the EBRT group. In high-risk patients, significantly more patients died from PC in the EBRT group than the RP group. Multivariate analysis demonstrated the RP group to be an independent prognostic factor for better CSS. On the other hand, the EBRT group had a significantly longer biochemical recurrence-free survival than the RP group.

Conclusions

Mortality outcomes of both RP and EBRT were generally favorable in low and intermediate risk patients. Improvement of CSS in high risk patients was seen in patients receiving RP over those receiving EBRT.