Effect of Antioxidant Flavonoids (Quercetin and Taxifolin) on In vitro Maturation of Porcine Oocytes.

Quercetin (QT) and taxifolin (TF) are structurally similar plant-derived flavonoids that have antioxidant properties and act as free radical scavengers. The objective of this study was to investigate effects of QT and TF on nuclear maturation of porcine oocytes. Effects of TF at 0, 1, 10, and 50 µg/mL on oocyte nuclear maturation (polar body extrusion) were investigated. After incubation for 44 h, there were no significant differences between the treatment and control groups except in the 50 µg/mL group which was significantly lower (59.2%, p<0.05) than the other groups (control: >80%). After parthenogenetic activation, further in vitro development of QT- or TF-treated vs control oocytes was investigated. A significantly higher proportion of QT-treated (1 µg/mL) oocytes developed into blastocysts compared to controls (24.3% vs 16.8%, respectively); however, cleavage rate and blastocyst cell number were not affected. The TF-treated group was not significantly different from controls. Levels of reactive oxygen species (ROS) and intracellular glutathione (GSH) in oocytes and embryos in a culture medium supplemented with QT or TF were measured. Both treatment groups had significantly lower (p<0.05) levels of ROS than controls, however GSH levels were different only in QT-treated oocytes. We conclude that exogenous flavonoids such as QT and TF reduce ROS levels in oocytes. Although at high concentration (50 µg/mL) both QT and TF appear to be toxic to oocytes.

Caffeic acid phenethyl ester lessens disease symptoms in an experimental autoimmune uveoretinitis mouse model.

Experimental autoimmune uveoretinitis (EAU) is an autoimmune disease that models human uveitis. Caffeic acid phenethyl ester (CAPE), a phenolic compound isolated from propolis, possesses anti-inflammatory and immunomodulatory properties. CAPE demonstrates therapeutic potential in several animal disease models through its ability to inhibit NF-κB activity. To evaluate these therapeutic effects in EAU, we administered CAPE in a model of EAU that develops after immunization with interphotoreceptor retinal-binding protein (IRBP) in B10.RIII and C57BL/6 mice. Importantly, we found that CAPE lessened the severity of EAU symptoms in both mouse strains. Notably, treated mice exhibited a decrease in the ocular infiltration of immune cell populations into the retina; reduced TNF-α, IL-6, and IFN-γ serum levels: and inhibited TNF-α mRNA expression in retinal tissues. Although CAPE failed to inhibit IRBP-specific T cell proliferation, it was sufficient to suppress cytokine, chemokine, and IRBP-specific antibody production. In addition, retinal tissues isolated from CAPE-treated EAU mice revealed a decrease in NF-κB p65 and phospho-IκBα. The data identify CAPE as a potential therapeutic agent for autoimmune uveitis that acts by inhibiting cellular infiltration into the retina, reducing the levels of pro-inflammatory cytokines, chemokine, and IRBP-specific antibody and blocking NF-κB pathway activation.

Oct4 overexpression facilitates proliferation of porcine fibroblasts and development of cloned embryos.

Octamer-binding transcription factor 4 (Oct4) is a critical molecule for the self-renewal and pluripotency of embryonic stem cells. Recent reports have shown that Oct4 also controls cell-cycle progression and enhances the proliferation of various types of cells. As the high proliferation of donor fibroblasts is critical to the production of transgenic pigs, using the somatic cell nuclear transfer technique, we analysed the effect of Oct4 overexpression on the proliferation of porcine fibroblasts and embryos. Porcine endogenous Oct4 cDNA was cloned, sequenced and inserted into an expression vector. The vector was transfected into porcine fibroblasts, and a stable Oct4-overexpressed cell line was established by antibiotic selection. Oct4 expression was validated by the immunostaining of Oct4. Cell morphology was changed to sharp, and both proliferation and migration abilities were enhanced in Oct4-overexpressed cells. Real-time RT-PCR results showed that p16, Bcl2 and Myc were upregulated in Oct4-overexpressed cells. Somatic cell nuclear transfer was performed using Oct4-overexpressed cells, and the development of Oct4 embryos was compared with that of wild-type cloned embryos. The cleavage and blastocyst formation rates were improved in the Oct4 embryos. Interestingly, blastocyst formation of the Oct4 embryos was observed as early as day 5 in culture, while blastocysts were observed from day 6 in wild-type cloned embryos. In conclusion, the overexpression of Oct4 enhanced the proliferation of both porcine fibroblasts and embryos.

Production and characterization of soluble human TNFRI-Fc and human HO-1(HMOX1) transgenic pigs by using the F2A peptide.

Generation of transgenic pigs for xenotransplantation is one of the most promising technologies for resolving organ shortages. Human heme oxygenase-1 (hHO-1/HMOX1) can protect transplanted organs by its strong anti-oxidative, anti-apoptotic, and anti-inflammatory effects. Soluble human TNFRI-Fc (shTNFRI-Fc) can inhibit the binding of human TNF-α (hTNF-α) to TNF receptors on porcine cells, and thereby, prevent hTNF-α-mediated inflammation and apoptosis. Herein, we successfully generated shTNFRI-Fc-F2A-HA-hHO-1 transgenic (TG) pigs expressing both shTNFRI-Fc and hemagglutinin-tagged-human heme oxygenase-1 (HA-hHO-1) by using an F2A self-cleaving peptide. shTNFRI-Fc and HA-hHO-1 transgenes containing the F2A peptide were constructed under the control of the CAG promoter. Transgene insertion and copy number in the genome of transgenic pigs was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. Expressions of shTNFRI-Fc and HA-hHO-1 in TG pigs were confirmed using PCR, RT-PCR, western blot, ELISA, and immunohistochemistry. shTNFRI-Fc and HA-hHO-1 were expressed in various organs, including the heart, lung, and spleen. ELISA assays detected shTNFRI-Fc in the sera of TG pigs. For functional analysis, fibroblasts isolated from a shTNFRI-Fc-F2A-HA-hHO-1 TG pig (i.e., #14; 1 × 10(5) cells) were cultured with hTNF-α (20 ng/mL) and cycloheximide (10 µg/mL). The viability of shTNFRI-Fc-F2A-HA-hHO-1 TG pig fibroblasts was significantly higher than that of the wild type (wild type vs. shTNFRI-Fc-F2A-HA-hHO-1 TG at 24 h, 31.6 ± 3.2 vs. 60.4 ± 8.3 %, respectively; p < 0.05). Caspase-3/-7 activity of the shTNFRI-Fc-F2A-HA-hHO-1 TG pig fibroblasts was lower than that of the wild type pig fibroblasts (wild type vs. shTNFRI-Fc-F2A-HA-hHO-1 TG at 12 h, 812,452 ± 113,078 RLU vs. 88,240 ± 10,438 RLU, respectively; p < 0.05). These results show that shTNFRI-Fc and HA-hHO-1 TG pigs generated by the F2A self-cleaving peptide express both shTNFRI-Fc and HA-hHO-1 molecules, which provides protection against oxidative and inflammatory injury. Utilization of the F2A self-cleaving peptide is a promising tool for generating multiple TG pigs for xenotransplantation.

Spirosoma radiotolerans sp. nov., a gamma-radiation-resistant bacterium isolated from gamma ray-irradiated soil.

A Gram-negative, short-rod-shaped bacterial strain with gliding motility, designated as DG5A(T), was isolated from a rice field soil in South Korea. Phylogenic analysis using 16S rRNA gene sequence of the new isolate showed that strain DG5A(T) belong to the genus Spirosoma in the family Spirosomaceae, and the highest sequence similarities were 95.5 % with Spirosoma linguale DSM 74(T), 93.4 % with Spirosoma rigui WPCB118(T), 92.8 % with Spirosoma luteum SPM-10(T), 92.7 % with Spirosoma spitsbergense SPM-9(T), and 91.9 % with Spirosoma panaciterrae Gsoil 1519(T). Strain DG5A(T) revealed resistance to gamma and UV radiation. Chemotaxonomic data showed that the most abundant fatty acids were summed feature C(16:1) ω7c/C(16:1) ω6c (36.90 %), C(16:1) ω5c (29.55 %), and iso-C(15:0) (14.78 %), and the major polar lipid was phosphatidylethanolamine (PE). The DNA G+C content of strain DG5A(T) was 49.1 mol%. Together, the phenotypic, phylogenetic, and chemotaxonomic data supported that strain DG5A(T) presents a novel species of the genus Spirosoma, for which the name Spirosoma radiotolerans sp. nov., is proposed. The type strain is DG5A(T) (=KCTC 32455(T) = JCM19447(T)).

Replacement of glutamine with the dipeptide derivative alanyl-glutamine enhances in vitro maturation of porcine oocytes and development of embryos.

The presence of glutamine (Gln) in in vitro maturation (IVM) and in vitro culture (IVC) medium is a more potent factor for improving porcine oocyte and embryo development than other amino acids. However Gln is inherently unstable and spontaneously breaks down into ammonia, and therefore interferes with proper development. To avoid this adverse effect, Gln was replaced in the present study with its stable dipeptide derivative alanyl-glutamine (Ala-Gln) and the effects of this replacement on porcine IVM and IVC were evaluated. Replacement of Gln with Ala-Gln during IVM did not improve nuclear maturation, however numbers of early cleaved embryos were significantly increased after activation. Blastocyst formation rates were also significantly improved by using Ala-Gln during IVM. Replacement of Gln with Ala-Gln during IVC significantly increased total cell numbers in blastocysts. Blastocyst formation rate was also significantly higher when Ala-Gln was used in both IVM and IVC. In conclusion, the use of Ala-Gln rather than Gln gives better results for development in both porcine IVM and IVC.

Production of Mutated Porcine Embryos Using Zinc Finger Nucleases and a Reporter-based Cell Enrichment System.

To facilitate the construction of genetically-modified pigs, we produced cloned embryos derived from porcine fibroblasts transfected with a pair of engineered zinc finger nuclease (ZFN) plasmids to create targeted mutations and enriched using a reporter plasmid system. The reporter expresses RFP and eGFP simultaneously when ZFN-mediated site-specific mutations occur. Thus, double positive cells (RFP(+)/eGFP(+)) were selected and used for somatic cell nuclear transfer. Two types of reporter based enrichment systems were used in this study; the cloned embryos derived from cells enriched using a magnetic sorting-based system showed better developmental competence than did those derived from cells enriched by flow cytometry. Mutated sequences, such as insertions, deletions, or substitutions, together with the wild-type sequence, were found in the cloned porcine blastocysts. Therefore, genetic mutations can be achieved in cloned porcine embryos reconstructed with ZFN-treated cells that were enriched by a reporter-based system.

Effect of 7,8-dihydroxyflavone as an antioxidant on in vitro maturation of oocytes and development of parthenogenetic embryos in pigs.

One of the factors that impairs in vitro produced porcine embryos is the oxidative stress that is mainly caused by the imbalance between reactive oxygen species (ROS) generation and antioxidants activity, especially that of glutathione (GSH). Here, we examined the effect of 7,8-dihydroxyflavone (7,8-DHF), a kind of flavonoid antioxidant, on porcine oocyte maturation and its developmental competence. Porcine oocytes were cultured in media supplemented with 0, 1, 5 and 10 µM 7,8-DHF during both in vitro maturation (IVM) and in vitro culture (IVC) after parthenogenetic activation. Maturation of oocytes was evaluated based on first polar body (PB) extrusion and intracellular GSH level, and developmental competence was assessed through observing cleavage and blastocyst formation. In each step, the levels of intracellular GSH and ROS were assessed by fluorescence intensity, and the apoptosis-related gene expression was examined using semiquantitative RT-PCR. The group treated with 1 µM 7,8-DHF during IVM and IVC showed increased cytoplasmic maturation and reached the blastocysts stage (36.1%) at a higher rate than the other groups (24.7, 16.0 and 10.3% for 0, 5 and 10 µM, P<0.05). In that group, the intracellular GSH level was significantly increased while ROS generation was significantly decreased after IVM and IVC (P<0.05). Moreover, it showed high expression of an anti-apoptotic gene (BCL2L1) and low expression of a pro-apoptotic gene (BAK1) (P<0.05). In conclusion, treatment with 1 µM 7,8-DHF during IVM and IVC showed an anti-apoptotic effect by increasing intracellular GSH synthesis and scavenging ROS and therefore improved the developmental competence of porcine embryos.

Photocrosslinkable natural polymers in tissue engineering.

Natural polymers have been widely used in scaffolds for tissue engineering due to their superior biocompatibility, biodegradability, and low cytotoxicity compared to synthetic polymers. Despite these advantages, there remain drawbacks such as unsatisfying mechanical properties or low processability, which hinder natural tissue substitution. Several non-covalent or covalent crosslinking methods induced by chemicals, temperatures, pH, or light sources have been suggested to overcome these limitations. Among them, light-assisted crosslinking has been considered as a promising strategy for fabricating microstructures of scaffolds. This is due to the merits of non-invasiveness, relatively high crosslinking efficiency via light penetration, and easily controllable parameters, including light intensity or exposure time. This review focuses on photo-reactive moieties and their reaction mechanisms, which are widely exploited along with natural polymer and its tissue engineering applications.

Paradoxical effects of kisspeptin: it enhances oocyte in vitro maturation but has an adverse impact on hatched blastocysts during in vitro culture.

Kisspeptin (Kp) is best known as a multifunctional peptide with roles in reproduction, the cardiovascular system and cancer. In the present study the expression of kisspeptin hierarchy elements (KISS1, GNRH1 and LHB) and their receptors (KISS1R, GNRHR and LHCGR, respectively) in porcine ovary and in cumulus-oocyte complexes (COCs) were investigated, as were its effects on the in vitro maturation (IVM) of oocytes and their subsequent ability to sustain preimplantation embryo competence after parthenogenetic electrical activation. Kp system elements were expressed and affected IVM of oocytes when maturation medium was supplemented with 10(-6)M Kp. Oocyte maturation, maternal gene expression (MOS, GDF9 and BMP15), blastocyst formation rate, blastocyst hatching and blastocyst total cell count were all significantly increased when oocytes were matured in medium containing Kp compared with the control group (without Kp). A Kp antagonist (p234) at 4×10(-6)M interfered with this hierarchy but did not influence the threshold effect of gonadotrophins on oocyte maturation. FSH was critical and permissive to Kp action on COCs by increasing the relative expression of KISS1R. In contrast, Kp significantly increased apoptosis, the expression of pro-apoptotic gene, BAK1, and suppressed trophoblast outgrowths from hatched blastocysts cultured on feeder cells. The present study provides the first functional evidence of the Kp hierarchy in porcine COCs and its role in enhancing oocyte maturation and subsequent developmental competence in an autocrine-paracrine manner. However, Kp supplementation may have a harmful impact on cultured hatched blastocysts reflecting systemic or local regulation during the critical early period of embryonic development.

Developmental competence of porcine oocytes after in vitro maturation and in vitro culture under different oxygen concentrations.

In this study, we investigated the effect of two oxygen concentrations (5 and 20%) during in vitro maturation (IVM) and during in vitro culture (IVC) on porcine embryo development and analysed differences in gene expression between cumulus-oocyte complexes matured under 5 or 20% oxygen and the resulting blastocysts cultured under 5% or 20% oxygen following parthenogenetic activation. There was no significant difference in oocyte maturation rate. However, the numbers of resulting blastocysts were significantly increased in the 5% IVC group compared with the 20% IVC group. Moreover, the M20C5 treatment group (23.01%) supported greater blastocyst development compared with the M5C5 (14.32%), M5C20 (10.30%), and M20C20 (17.88%) groups. However, total cell numbers were not significantly different among groups. According to mRNA abundance data of multiple genes, each treatment altered the expression of genes in different patterns. GLUT1, G6PD and LDHA were up-regulated in cumulus cells that had been matured in low oxygen, suggesting a higher glucose uptake and an increase in anaerobic glycolysis, whereas cyclin B1 (CCNB) and MnSOD (Mn-superoxide dismutase) were upregulated in cumulus cells that had been matured in high oxygen, which suggests a higher activity of mitosis-promoting factor and antioxidant response. In spite of these differential effects on cumulus cells, oocytes could mature normally regardless of different oxygen concentrations. Therefore, it can be concluded that high oxygen concentration during in vitro maturation and low oxygen during in vitro culture may alter the expression of multiple genes related to oocyte competence and significantly improves embryo development (p < 0.05) but not blastocyst quality.

Effect of oocyte-secreted factors on porcine in vitro maturation, cumulus expansion and developmental competence of parthenotes.

The oocyte is known from recent studies in the mouse, cow, sheep and human to be a central regulator of follicular cell function. However, in the pig, little information is known about the regulation of cumulus expansion by oocyte-secreted factors and oocyte quality. We investigated the possible effects of oocyte-secreted factors during in vitro maturation on cumulus expansion and on porcine oocytes as judged by subsequent embryonic development after parthenogenetic activation. Cumulus-oocyte complexes (COC) from antral follicles of pig ovaries collected from a local abattoir were divided into control and treatment groups and were cultured in tissue culture medium 199 supplemented with follicle-stimulating hormone. Treatment groups consisted of increasing numbers of denuded oocytes (DO) co-cultured with COC (at ratios of COC to DO of 1:1, 1:2, 1:3, 1:4 and 1:5). After incubation for 44 h, cumulus expansion and maturation rates were assessed and oocytes were activated parthenogenetically. Cumulus expansion in the 1 COC:4 DO and 1 COC:5 DO groups was low and altered because full dispersion of the outer layer did not occur. Cell viability was not affected, as measured by the automated cell counter, but scanning electron microscopy revealed only a scanty extracellular matrix. Blastocyst rate was significantly higher in the 1 COC:4 DO (34.4%) and in the 1 COC:5 DO (34.9%) groups (p < 0.05) when compared with other groups. Maturation rate, cleavage rate and total cell number showed no significant difference between control and treatment groups. Amplification by reverse transcription polymerase chain reaction (RT-PCR) showed up-regulation of growth differentiation factor 9 (GDF9) in the cumulus cells in the 1 COC:4 DO group at 44 h. We conclude that denuded porcine oocytes could improve the maturation of COC as evidenced by increased blastocyst development in the 1 COC:4 DO, even though cumulus expansion was poor. This improvement could be a result of the GDF9 up-regulation.

The 9-cis retinoic acid signaling pathway and its regulation of prostaglandin-endoperoxide synthase 2 during in vitro maturation of pig cumulus cell-oocyte complexes and effects on parthenogenetic embryo production.

The addition of 9-cis retinoic acid to the oocyte maturation culture medium has a beneficial effect on in vitro fertilized embryos. However, the mechanism of this activity is not known. Therefore, this study was done to elucidate the effect of 9-cis retinoic acid on parthenogenetic embryo production and its signaling pathway and molecular function during in vitro maturation of porcine cumulus cell-oocyte complexes (COCs). Concentrations of 0, 5, 50, and 500 nM 9-cis retinoic acid were added to the in vitro maturation medium, and the embryos were assessed after parthenogenetic activation. Cumulus cells and oocytes from the in vitro matured COCs were separated and subjected to RT-PCR and real-time RT-PCR for detecting retinoic acid receptors and measuring expression of prostaglandin-endoperoxide synthase1 and 2. The addition of 5 nM 9-cis retinoic acid to the maturation medium was beneficial for parthenogenetic embryo production. The effect of 9-cis retinoic acid was exerted directly through the oocytes via the retinoic acid receptor alpha and retinoid X receptor gamma signaling pathways and indirectly through the cumulus cells by the retinoic acid receptor beta and gamma and retinoid X receptor alpha and beta signaling pathways. The addition of 5 nM 9-cis retinoic acid-stimulated cumulus cells reaches full expansion by suppressing their excessive expression of prostaglandin-endoperoxide synthase 2. This study shows that 9-cis retinoic acid can exert its beneficial effect on parthenogenetic embryo production in pigs by multidimensional pathways affecting oocyte maturation.

Short-term treatment with 6-DMAP and demecolcine improves developmental competence of electrically or Thi/DTT-activated porcine parthenogenetic embryos.

Treatment with 6-dimethylaminopurine (6-DMAP) or demecolcine (DE) for several (at least 2) hours after artificial activation is known to improve in vitro development of porcine embryos. However, several reports have also shown that treatments with these chemicals induce apoptosis. The aim of this study was to find out whether short-term treatment with 6-DMAP and DE combined with electrical or thimerosal/dithiothreitol (Thi/DTT) activation had a beneficial effect on development of parthenogenetically activated porcine oocytes. We additionally treated embryos with 6-DMAP (2 mM) and/or DE (0.4 microg/ml) for a short time (40 min) after an electrical pulse (EP) or Thi/DTT. As a result, short-term treatment with 6-DMAP and DE successfully induced development of electrically or Thi/DTT-activated porcine parthenogenetic embryos with no significant difference in cleavage rate, blastocyst formation rate and total cell number compared with long-term treatment. To find optimal activation protocol, cleavage rate, blastocyst formation rate and total cell number were compared between EP and Thi/DTT treatments. Thi/DTT + 6-DMAP + DE showed significantly higher blastocyst formation rate (36.1 ± 3.5%) and total cell number (46.9 ± 1.0) than other groups (EP + 6-DMAP + DE, EP + Thi/DTT + 6-DMAP + DE: 23.3 ± 3.0%, 42.2 ± 1.1 and 17.2 ± 2.7%, 36.7 ± 1.5, respectively). In conclusion, this study demonstrates that short-term treatment with 6-DMAP and DE is as effective as the standard long-term treatment and Thi/DTT + 6-DMAP + DE exerts a synergistic effect.

Blastocysts derived from adult fibroblasts of a rhesus monkey ( Macaca mulatta) using interspecies somatic cell nuclear transfer.

In non-human primates, it is difficult to collect sufficient numbers of oocytes for producing identical embryos by somatic cell nuclear transfer (SCNT). Because of this factor, inter-species SCNT (iSCNT) using heterospecific oocytes is an attractive alternative approach. The objective of this study was to produce iSCNT-derived blastocysts using enucleated cow (Bos taurus) metaphase II oocytes and adult rhesus monkey (Macaca mulatta) fibroblasts. Ear skin tissue from a 6-year-old male rhesus monkey was collected by biopsy and fibroblasts were isolated. Immature cumulus-oocyte complexes from cow ovaries were collected and matured in vitro in Medium 199. The enucleated oocytes were reconstructed with rhesus monkey fibroblasts and iSCNT embryos were cultured in modified synthetic oviduct fluid in an atmosphere of 5-5.5% CO2 under various conditions (37-39 °C and 5-20% O2) to examine the effects of in vitro culture conditions. Most embryos were arrested at the 8- or 16-cell stage and only three blastocysts were derived in this way using iSCNT from a total of 1153 cultured activated embryos (0.26% production rate). Two of the three blastocysts were used for counting nuclear numbers using bisbenzimide staining, which were 51 and 24. The other iSCNT-derived blastocyst was used to analyse mitochondrial DNA (mtDNA) by PCR, and both rhesus monkey and cow mtDNA were detected. Although the development rate was extremely low, this study established that iSCNT using two phylogenetically distant species, including a primate, could produce blastocysts. With improvements in the development rate, it may be possible to produce rhesus monkey iSCNT-derived embryonic stem cell lines for studies on primate nucleus and cow mitochondria interaction mechanisms.

Effects of vitamin D supplements in patients with chronic hepatitis C: a randomized, multi-center, open label study.

BACKGROUND/AIMS: We aimed to assess the role of vitamin D supplementation in the response to pegylated interferon-α (PEG-IFN-α) plus ribavirin (RBV) treatment in patients with chronic hepatitis C (CHC). METHODS: Our study was a multi-center, randomized controlled trial in 11 hospitals. CHC patients were randomly assigned (1:1) to two groups namely, PEGIFN-α plus RBV (control group) or PEG-IFN-α plus RBV + vitamin D (800 IU daily) (vitamin D group). The primary end-point was the rate of sustained virologic response (SVR). RESULTS: One hundred forty eight CHC patients were randomly assigned to two groups. Seventy-one patients received the PEG-IFN-α plus RBV and 77 patients received the PEG-IFN-α plus RBV + vitamin D. A total of 105 patients completed the study (control group, 47 vs. vitamin D group, 58). Baseline characteristics were mostly similar in both the groups. There was a modest but non-significant increase in SVR in the vitamin D group compared to the control group with the intention to treat analysis (64.0% vs. 49.3 %, p = 0.071) as well as in the per protocol analysis (control group vs. vitamin D group: 74.5% vs. 84.5%, p = 0.202). Fifty-two patients (73.2%) in the control group and 63 patients (81.8%) in the vitamin D group experienced at least one adverse event. The drop-out rate due to adverse effects was not different between both groups (control group vs. vitamin D group: 19.7% vs. 10.4%, p = 0.111). CONCLUSION: Vitamin D supplement did not increase SVR in treatment naïve patients with CHC irrespective of genotype.

Efficacy of Pegylated Interferon Monotherapy versus Sequential Therapy of Entecavir and Pegylated Interferon in Hepatitis B e Antigen-Positive Hepatitis B Patients: A Randomized, Multicenter, Phase IIIb Open-Label Study (POTENT Study).

BACKGROUND: Until now, various types of combined therapy with nucleotide analogs and pegylated interferon (Peg-INF) in patients with hepatitis B patients have been tried. However, studies regarding the benefits of de novo combination, late-add on, and sequential treatment are very limited. The objective of the current study was to identify the efficacy of sequential treatment of Peg-INF after short-term antiviral treatment. METHODS: Between June 2010 and June 2015, hepatitis B e antigen (HBeAg)-positive patients (n = 162) received Peg-IFN for 48 weeks (mono-treatment group, n = 81) and entecavir (ETV) for 12 weeks with a 48-week course of Peg-IFN starting at week 5 of ETV therapy (sequential treatment group, n = 81). The primary endpoint was HBeAg seroconversion at the end of follow-up period after the 24-week treatment. The primary endpoint was analyzed using Chi-square test, Fisher's exact test, and regression analysis. RESULTS: HBeAg seroconversion rate (18.2% vs. 18.2%, t = 0.03, P = 1.000) and seroclearance rate (19.7% vs. 19.7%, t = 0.03, P = 1.000) were same in both mono-treatment and sequential treatment groups. The rate of alanine aminotransferase (ALT) normalization (45.5% vs. 54.5%, t = 1.12, P = 0.296) and serum hepatitis B virus (HBV)-DNA <2000 U/L (28.8% vs. 28.8%, t = 0.10, P = 1.000) was not different in sequential and mono-treatment groups at 24 weeks of Peg-INF. Viral response rate (HBeAg seroconversion and serum HBV-DNA <2000 U/L) was not different in the two groups (12.1% vs. 16.7%, t = 1.83, P = 0.457). Baseline HBV-DNA level (7 log10U/ml vs. 7.5 log10U/ml, t = 1.70, P = 0.019) and hepatitis B surface antigen titer (3.6 log10U/ml vs. 4.0 log10U/ml, t = 2.19, P = 0.020) were lower and predictors of responder in mono-treatment and sequential treatment groups, respectively. CONCLUSIONS: The current study shows no differences in HBeAg seroconversion rate, ALT normalization, and HBV-DNA levels between mono-therapy and sequential therapy regimens. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01220596; https://clinicaltrials.gov/ct2/show/NCT01220596?term=NCT01220596&rank=1.

Soluble expression and purification of receptor activator of nuclear factor-kappa B ligand using Escherichia coli.

Receptor activator of nuclear factor-kappa B ligand (RANKL) is a critical factor in osteoclastogenesis. It makes osteoclasts differentiate and multinucleate in bone remodeling. In the present study, RANKL was expressed as a soluble maltose binding protein (MBP)-fusion protein using the Escherichia coli maltose binding domain tag system (pMAL) expression vector system. The host cell E. coli DH5α was cultured and induced by isopropyl ß-D-1- thiogalactopyranoside for rRANKL expression. Cells were disrupted by sonication to collect soluble MBP-fused rRANKL. The MBP-fusion rRANKL was purified with MBP Trap affinity chromatography and treated with Tobacco Etch Virus nuclear inclusion endopeptidase (TEV protease) to remove the MBP fusion protein. Dialysis was then carried out to remove binding maltose from the cleaved rRANKL solution. The cleaved rRANKL was purified with a second MBP Trap affinity chromatography to separate unsevered MBP-fusion rRANKL and cleaved MBP fusion protein. The purified rRANKL was shown to have biological activity by performing in vitro cell tests. In conclusion, biologically active rRANKL was successfully purified by a simple two-step chromatography purification process with one column.

Intrapancreatic ectopic splenic tissue found in a cloned miniature pig.

Somatic cell nuclear transfer (SCNT) is a cost-effective technique for producing transgenic pigs. However, abnormalities in the cloned pigs might prevent use these animals for clinical applications or disease modeling. In the present study, we generated several cloned pigs. One of the pigs was found to have intrapancreatic ectopic splenic tissue during histopathology analysis although this animal was grossly normal and genetically identical to the other cloned pigs. Ectopic splenic tissue in the pancreas is very rare, especially in animals. To the best of our knowledge, this is the first such report for cloned pigs.

Relationship between pregnancy rate and serum progesterone concentration in cases of porcine embryo transfer.

The level of P4 at the time of embryo transfer (ET) is important. P4 concentrations and numbers of corpora lutea for 126 recipients were evaluated. Nuclear transfer embryos were transferred into 126 surrogates. 11 maintained their pregnancy until full-term delivery, 17 miscarried, and implantation failed in 98 animals. P4 levels in the full-term group were significantly different from those of the pigs that aborted or in which implantation failed (p < 0.05). However, the numbers of corpora lutea were not significantly different. These findings indicate that the concentration of progesterone can be an important factor for successful ET in pigs.