HAS3-induced accumulation of hyaluronan in 3D MDCK cultures results in mitotic spindle misorientation and disturbed organization of epithelium

The amount of hyaluronan (HA) is low in simple epithelia under normal conditions, but during tumorigenesis, trauma or inflammation HA is increased on the epithelial cells and surrounding stroma. Excessive HA in epithelia is suggested to interfere with cell–cell adhesions, resulting in disruption of the epithelial barrier function. In addition, stimulated HA synthesis has been correlated with epithelial-to-mesenchymal transition and invasion of cancer cells. However, the effects of HA overload on normal epithelial morphogenesis have not been characterized in detail. Madin-Darby canine kidney (MDCK) cells form polarized epithelial cysts, when grown in a 3-dimensional (3D) matrix. These cells were used to investigate whether stimulated HA synthesis, induced by stable overexpression of GFP-HAS3, influences cell polarization and epithelial morphogenesis. GFP-HAS3 expression in polarized MDCK cells resulted in active HA secretion at apical and basolateral membrane domains. HA-deposits interfered with the formation of cell–cell junctions, resulting in impaired barrier function. In 3D cyst cultures, HA accumulated into apical lumina and was also secreted from the basal side. The HAS3-expressing cysts failed to form a single lumen and instead displayed multiple small lumina. This phenotype was correlated with aberrant mitotic spindle orientation in dividing cells. The results of this study indicate that excess pericellular HA disturbs the normal cell–cell and cell–ECM interactions in simple epithelia, leading to aberrant epithelial morphogenesis. The morphological abnormalities observed in 3D epithelial cultures upon stimulated HAS3 expression may be related to premalignant changes, including intraluminal invasion and deregulated epithelialization, probably mediated by the mitotic spindle orientation defects.     

Epithelial homeostasis: elimination by live cell extrusion

To maintain a functional and harmonious epithelial society, the number and quality of cells need to be tightly controlled. Two recent studies reveal a novel cellular process for epithelial homeostasis: crowding-mediated live cell extrusion.     

Discovery and preclinical profile of teneligliptin (3-[(2S,4S)-4-[4-(3-methyl-1-phenyl-1H-pyrazol-5-yl)piperazin-1-yl]pyrrolidin-2-ylcarbonyl]thiazolidine): A highly potent,selective, long-lasting and orally active dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes

Dipeptidyl peptidase IV (DPP-4) inhibition is suitable mechanism for once daily oral dosing regimen because of its low risk of hypoglycemia. We explored linked bicyclic heteroarylpiperazines substituted at the γ-position of the proline structure in the course of the investigation of l-prolylthiazolidines. The efforts led to the discovery of a highly potent, selective, long-lasting and orally active DPP-4 inhibitor, 3-[(2S,4S)-4-[4-(3-methyl-1-phenyl-1H-pyrazol-5-yl)piperazin-1-yl]pyrrolidin-2-ylcarbonyl]thiazolidine (8g), which has a unique structure characterized by five consecutive rings. An X-ray co-crystal structure of 8g in DPP-4 demonstrated that the key interaction between the phenyl ring on the pyrazole and the S₂ extensive subsite of DPP-4 not only boosted potency, but also increased selectivity. Compound 8g, at 0.03 mg/kg or higher doses, significantly inhibited the increase of plasma glucose levels after an oral glucose load in Zucker fatty rats. Compound 8g (teneligliptin) has been approved for the treatment of type 2 diabetes in Japan.     

Room temperature storage of mouse epididymal spermatozoa: exploration of factors affecting sperm survival

To explore optimal conditions for in vitro sperm survival, we examined the effects of several media used for murine egg culture and in vitro fertilization (IVF; including M16, M2, PB1, TYH, and CZB) on motility of murine spermatozoa stored at 22 degrees C under paraffin oil. Of media tested, M2 medium, that had been adjusted to pH 7.2 by adding N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), was found to be the best. Addition of various concentrations of HEPES to TYH did not improve sperm survival, suggesting that HEPES (and probably neutral pH) do not enhance survival of murine sperm. Since M16 has higher amounts of bicarbonate than M2 (25 mM versus 4.15 mM), four variations of M16 media containing 4.15, 8.30, 16.60, or 33.20 mM bicarbonate were prepared and tested. The modified M16 media with 4.15-16.60 mM bicarbonate yielded good sperm survival (comparable to M2 medium), while relatively high concentrations of bicarbonate (ranging from 16.60 to 33.20 mM) were deleterious to isolated sperm, suggesting the need for a minimum level of residual bicarbonate. However, the mechanism by which the lifespan of spermatozoa is extended remains unknown. The in vitro fertilizing abilities of spermatozoa left in M2 medium for 1, 3, and 5 days at 22 degrees C were 52.5, 21.8, and 7.0%, respectively, when the cleavage rate to the two-cell stage was examined. Transfer of two-cell embryos produced in vitro with spermatozoa stored for 1, 3, and 5 days at 22 degrees C resulted in production of fetuses with efficiencies of 42.5, 23.4, and 12.5%, respectively, which were lower than that of embryos derived from in vitro fertilization with fresh spermatozoa (68.1%). In conclusion, spermatozoa kept in M2 medium for up to 5 days at 22 degrees C can fertilize oocytes.     

Finite Adaptation and Multistep Moves in the Metropolis-Hastings Algorithm for Variable Selection in Genome-Wide Association Analysis

High-dimensional datasets with large amounts of redundant information are nowadays available for hypothesis-free exploration of scientific questions. A particular case is genome-wide association analysis, where variations in the genome are searched for effects on disease or other traits. Bayesian variable selection has been demonstrated as a possible analysis approach, which can account for the multifactorial nature of the genetic effects in a linear regression model.Yet, the computation presents a challenge and application to large-scale data is not routine. Here, we study aspects of the computation using the Metropolis-Hastings algorithm for the variable selection: finite adaptation of the proposal distributions, multistep moves for changing the inclusion state of multiple variables in a single proposal and multistep move size adaptation. We also experiment with a delayed rejection step for the multistep moves. Results on simulated and real data show increase in the sampling efficiency. We also demonstrate that with application specific proposals, the approach can overcome a specific mixing problem in real data with 3822 individuals and 1,051,811 single nucleotide polymorphisms and uncover a variant pair with synergistic effect on the studied trait. Moreover, we illustrate multimodality in the real dataset related to a restrictive prior distribution on the genetic effect sizes and advocate a more flexible alternative.     

Dominant Incidence of Multidrug and Extensively Drug-Resistant Specific Mycobacterium tuberculosis Clones in Osaka Prefecture, Japan

Infection and transmission of multidrug-resistant Mycobacterium tuberculosis (MDR-Mtb) and extensively drug-resistant M. tuberculosis (XDR-Mtb) is a serious health problem. We analyzed a total of 1,110 Mtb isolates in Osaka Prefecture and neighboring areas from April 2000 to March 2009. A total of 89 MDR-Mtb were identified, 36 (48.5%) of which were determined to be XDR-Mtb. Among the 89 MDR-Mtb isolates, 24 (27.0%) phylogenetically distributed into six clusters based on mycobacterial interspersed repetitive units-various number of tandem repeats (MIRU-VNTR) typing. Among these six clusters, the MIRU-VNTR patterns of four (OM-V02, OM-V03, OM-V04, and OM-V06) were only found for MDR-Mtb. Further analysis revealed that all isolates belonging to OM-V02 and OM-V03, and two isolates from OM-V04 were clonal. Importantly such genotypes were not observed for drug-sensitive isolates. These suggest that few but transmissible clones can transmit after acquiring multidrug resistance and colonize even in a country with a developed, well-organized healthcare system.     

Fabrication of Mouse Embryonic Stem Cell-Derived Layered Cardiac Cell Sheets Using a Bioreactor Culture System

Bioengineered functional cardiac tissue is expected to contribute to the repair of injured heart tissue. We previously developed cardiac cell sheets using mouse embryonic stem (mES) cell-derived cardiomyocytes, a system to generate an appropriate number of cardiomyocytes derived from ES cells and the underlying mechanisms remain elusive. In the present study, we established a cultivation system with suitable conditions for expansion and cardiac differentiation of mES cells by embryoid body formation using a three-dimensional bioreactor. Daily conventional medium exchanges failed to prevent lactate accumulation and pH decreases in the medium, which led to insufficient cell expansion and cardiac differentiation. Conversely, a continuous perfusion system maintained the lactate concentration and pH stability as well as increased the cell number by up to 300-fold of the seeding cell number and promoted cardiac differentiation after 10 days of differentiation. After a further 8 days of cultivation together with a purification step, around 1×10⁸ cardiomyocytes were collected in a 1-L bioreactor culture, and additional treatment with noggin and granulocyte colony stimulating factor increased the number of cardiomyocytes to around 5.5×10⁸. Co-culture of mES cell-derived cardiomyocytes with an appropriate number of primary cultured fibroblasts on temperature-responsive culture dishes enabled the formation of cardiac cell sheets and created layered-dense cardiac tissue. These findings suggest that this bioreactor system with appropriate medium might be capable of preparing cardiomyocytes for cell sheet-based cardiac tissue.     

Prostaglandin E2 promotes intestinal tumor growth via DNA methylation

Although aberrant DNA methylation is considered to be one of the key ways by which tumor-suppressor and DNA-repair genes are silenced during tumor initiation and progression, the mechanisms underlying DNA methylation alterations in cancer remain unclear. Here we show that prostaglandin E(2) (PGE(2)) silences certain tumor-suppressor and DNA-repair genes through DNA methylation to promote tumor growth. These findings uncover a previously unrecognized role for PGE(2) in the promotion of tumor progression.     

Effects of nitric oxide on mitochondrial permeability transition pore and thiol-mediated responses in cardiac myocytes

Nitric oxide (NO) alters the opening of mitochondrial permeability transition pore (mPTP). However, the signaling pathways of NO on mPTP remain elusive. We aimed to clarify the contribution of thiol-mediated responses to the effects of NO on mPTP in permeabilized myocytes. We found that (1) a high concentration of spermine NONOate (an NO donor; 500 μM) opened mPTP and depolarized ΔΨ(m). (2) A low concentration of NONOate (5 μM) prevented atractyloside (an mPTP opener)-induced mPTP opening. (3) Mn(III) tetrakis (4-benzoic acid) porphyrin (Mn-TBAP, ONOO(-) scavenger) attenuated the effect of high-concentration NONOate on mPTP opening, but did not inhibited the preventive effects of low-concentration NONOate. (4) When the interaction of NO with thiol was inhibited by N-ethylmaleimide, the opening (by high-concentration NONOate) and preventive effects (by low-concentration NONOate) of NONOate on mPTP were blocked. (5) Dithiothreitol (an inhibitor of disulfide bonds formation) prevented high-concentration NONOate-induced mPTP opening. (6) Ascorbic acid (an inhibitor of S-nitrosylation) prevented the preventive effects of low-concentration NONOate on mPTP. We conclude that opening of mPTP by high-concentration NO is related to disulfide bonds formation and oxidizing effects of ONOO(-). In contrast, the inhibitory effect of physiological concentrations of NO on mPTP is related to S-nitrosylation.