CDK inhibitor SU9516 induces tetraploid blastocyst formation from parthenogenetically activated porcine embryos

Objective

To examine the effect of SU9516, a cyclin-dependent kinase inhibitor, on the induction of tetraploid blastocyst formation in porcine embryos by parthenogenetic activation.

Results

Karyotype analysis of blastocysts showed that in the SU9516-treatment group 56% were tetraploid, whereas in the cytochalasin B (CB) group 67% were diploid. The level of maturation-promoting factor (MPF) in stimulated embryos treated with 10 µM SU9516 for 4 h was lower than in embryos treated with CB group (103 vs. 131 pg/ml). The mRNA expression levels of Nanog significantly increased in SU9516-treated embryos than CB group.

Conclusion

SU9516 can induce tetraploid blastocyst formation at high efficiency. SU9516 can significantly influence the in vitro developmental competence of porcine parthenogenetically activated embryos by influencing the level of MPF and the gene related apoptosis and pluripotency.

     

The developmental competence of oocytes parthenogenetically activated by an electric pulse and anisomycin treatment

Objective

The aim of this study was to investigate the developmental competence of oocytes parthenogenetically activated by an electric pulse (EP) and treated with anisomycin and to determine whether this method is applicable to somatic cell nuclear transfer (SCNT).

Results

Embryos derived from porcine oocytes parthenogenetically activated by an EP and treatment with 0.01 µg/mL anisomycin had a significantly improved in vitro developmental capacity. Furthermore, 66.6% of blastocysts derived from these embryos had a diploid karyotype. The blastocyst formation rate of cloned embryos was similar between oocytes activated by an EP and treated with 2 mM 6-dimethylaminopurine for 4 h and those activated by an EP and treated with 0.01 µg/mL anisomycin for 4 h. The level of maturation-promoting factor was significantly decreased in oocytes activated by an EP and treated with anisomycin. Finally, the mRNA expression levels of apoptosis-related genes (Bax and Bcl-2) and pluripotency-related genes (Oct4, Nanog, and Sox2) were checked by RT-PCR.

Conclusion

Our results demonstrate that porcine oocyte activation via an EP in combination with anisomycin treatment can lead to a high blastocyst formation rate in parthenogenetic activation and SCNT experiments.

     

Dynamic changes of histone H3 lysine 9 following trimethylation in bovine oocytes and pre-implantation embryos

Objectives

We have examined dynamic changes of histone H3 lysine 9 following trimethylation (H3K9me3), the mRNA expression levels of SUV39H1 and SUV39H2 in bovine oocytes and the role in the development of in vitro fertilization (IVF) pre-implantation embryos.

Results

There were strong H3K9me3 signals in germinal vesicle (GV) oocytes but no signals in MII oocytes. H3K9me3 signals were maintained during IVF pre-implantation embryo development. SUV39H1 and SUV39H2 showed significantly higher mRNA expression levels in GV oocytes than MII oocytes (P < 0.01). SUV39H1 showed high mRNA expression level in two-cell embryos, however, SUV39H2 showed high mRNA expression level in four-cell embryos. In other development stage, SUV39H1 and SUV39H2 showed low expression levels.

Conclusion

Bovine IVF pre-implantation embryos maintain strong H3K9me3 signals and SUV39H1 and SUV39H2 are highly expressed at the early development stage of pre-implantation embryos.

     

Noninvasive characterization of metabolites secreted in culture media by bovine embryos during in vitro production

Introduction

Secreted molecules could be correlated with the potential of embryonic development. The development of new technologies, such as mass spectrometry (MS), has enabled analyzes in culture medium to favor the determination of embryos viability in order to improve embryo selection.

Objectives

To perform a non-invasive characterization of the secretome of in vitro produced embryos with different kinetics of cleavage and in different stages of development to obtain specific patterns based on embryonic phenotype through MALDI–TOF–MS.

Methods

Bovine embryos were produced in vitro by standard protocols. The zygotes were transferred to individual culture medium and divided into two groups: Fast [4 cells-22 hours past the beginning of culture (hpc)] and Slow (2 cells-22 hpc). Culture media drops were collected at 22, 96 and 168 hpc. Analysis of embryonic secretome was made by MALDI–TOF–MS after extractions of the metabolites. Spectra were acquired in positive ionization mode. Univariate (Fold-change) and multivariate (Partial Least Squares Discriminants Analysis) analyses were performed by the online software Metaboanalyst.

Results

It was demonstrated that embryos with different kinetics have different spectrometric profiles during embryonic development. Moreover, secreted molecules in each developmental stage are differentially represented in embryos with different kinetics, and are related to specific pathways such as lipid and amino acids metabolism and cell proliferation.

Conclusion

We propose that the analysis of culture media by MALDI–TOF–MS can be used for qualitative characterization of bovine embryos, allowing the identification of key molecules during in vitro culture.

     

Corneal Collagen Cross-Linking for the Management of Mycotic Keratitis

Purpose

To evaluate the efficiency of corneal collagen cross-linking (CXL) in addition to topical voriconazole in cases with mycotic keratitis.

Design

Retrospective case series in a tertiary university hospital.

Participants

CXL was performed on 13 patients with mycotic keratitis who presented poor or no response to topical voriconazole treatment.

Methods

The clinical features, symptoms, treatment results and complications were recorded retrospectively. The corneal infection was graded according to the depth of infection into the stroma (from grade 1 to grade 3). The visual analogue scale was used to calculate the pain score before and 2 days after surgery.

Main Outcome Measures

Grade of the corneal infection.

Results

Mean age of 13 patients (6 female and 7 male) was 42.4 ± 17.7 years (20–74 years). Fungus was demonstrated in culture (eight patients) or cytological examination (five patients). Seven of the 13 patients (54%) were healed with topical voriconazole and CXL adjuvant treatment in 26 ± 10 days (15–40 days). The remaining six patients did not respond to CXL treatment; they initially presented with higher grade ulcers. Pre- and post-operative pain score values were 8 ± 0.8 and 3.5 ± 1, respectively (p < 0.05).

Conclusions

The current study suggests that adjunctive CXL treatment is effective in patients with small and superficial mycotic ulcers. These observations require further research by large randomized clinical trials.

     

SV40 Large T Antigen Disrupts Embryogenesis of Canine and Porcine Somatic Cell Nuclear Transfer Embryo

Background

Somatic cell nuclear transfer (SCNT) is a useful biotechnological tool for transgenic animal production using genetically modified somatic cells (GMSCs). However, there are several limitations preventing successful transgenic animal generation by SCNT, such as obtaining proper somatic donor cells with a sufficiently long life span and proliferative capacity for generating GMSCs. Here, we established simian virus 40 large T antigen (SV40LT)-mediated lifespan-extended canine fibroblast cells (SV40LT-K9 cells) and evaluated their potential as nuclei donors for SCNT, based on cellular integrity and SCNT embryo development.

Results

SV40LT did not cause canine cell transformation, based on cell morphology and proliferation rate. No anchorage-independent growth in vitro and tumorigenicity in vivo were observed. After SCNT with SV40LT-K9 cells, embryos were transferred into surrogate dogs. All dogs failed to become pregnant. Most embryos did not proceed past the 8-cell stage and only one surrogate showed an implantation trace in its oviduct, indicating that the cells rarely developed into blastocysts. Because of the absence of an in vitro maturation method for canine embryos, we performed identical experiments using porcine fibroblast cells. Similarly, SV40LT did not transform porcine fibroblast cells (SV40LT-Pig cells). During in vitro development of SV40LT-Pig cell-driven SCNT embryos, their blastocyst formation rate was clearly lower than those of normal cells. Karyotyping analysis revealed that both SV40LT-K9 and SV40LT-Pig cells had aberrant chromosomal statuses.

Conclusions

Although lifespan-extended canine and porcine cells via SV40LT exhibit no apparent transforming changes, they are inappropriate for use as nuclei donors for SCNT because of their aneuploidy.

     

RepSox improves viability and regulates gene expression in rhesus monkey–pig interspecies cloned embryos

Objective

To investigate the effect of the small molecule, RepSox, on the expression of developmentally important genes and the pre-implantation development of rhesus monkey–pig interspecies somatic cell nuclear transfer (iSCNT) embryos.

Results

Rhesus monkey cells expressing the monomeric red fluorescent protein 1 which have a normal (42) chromosome complement, were used as donor cells to generate iSCNT embryos. RepSox increased the expression levels of the pluripotency-related genes, Oct4 and Nanog (p < 0.05), but not of Sox2 compared with untreated embryos at the 2–4-cell stage. Expression of the anti-apoptotic gene, Bcl2, and the pro-apoptotic gene Bax was also affected at the 2–4-cell stage. RepSox treatment also increased the immunostaining intensity of Oct4 at the blastocyst stage (p < 0.05). Although the blastocyst developmental rate was higher in the group treated with 25 µM RepSox for 24 h than in the untreated control group (2.4 vs. 1.2%, p > 0.05), this was not significant.

Conclusion

RepSox can improve the developmental potential of rhesus monkey–pig iSCNT embryos by regulating the expression of pluripotency-related genes.

     

Optimization of fecal sample preparation for untargeted LC-HRMS based metabolomics

Introduction

Collecting feces is easy. It offers direct outcome to endogenous and microbial metabolites.

Objectives

In a context of lack of consensus about fecal sample preparation, especially in animal species, we developed a robust protocol allowing untargeted LC-HRMS fingerprinting.

Methods

The conditions of extraction (quantity, preparation, solvents, dilutions) were investigated in bovine feces.

Results

A rapid and simple protocol involving feces extraction with methanol (1/3, M/V) followed by centrifugation and a step filtration (10 kDa) was developed.

Conclusion

The workflow generated repeatable and informative fingerprints for robust metabolome characterization.

     

Minimal variation of the plasma lipidome after delayed processing of neonatal cord blood

Background

Cord blood lipids are potential disease biomarkers. We aimed to determine if their concentrations were affected by delayed blood processing.

Method

Refrigerated cord blood from six healthy newborns was centrifuged every 12 h for 4 days. Plasma lipids were analysed by liquid chromatography/mass spectroscopy.

Results

Of 262 lipids identified, only eight varied significantly over time. These comprised three dihexosylceramides, two phosphatidylserines and two phosphatidylethanolamines whose relative concentrations increased and one sphingomyelin that decreased.

Conclusion

Delay in separation of plasma from refrigerated cord blood has minimal effect overall on the plasma lipidome.

     

PEGylation of cytochrome <Emphasis Type="Italic">c</Emphasis> at the level of lysine residues mediated by a microbial <Emphasis Type="Italic">trans</Emphasis>glutaminase

Objectives

To establish a method for microbial transglutaminase (mTG)-mediated PEGylation of proteins at the level of lysine (Lys) residues.

Results

Carboxybenzyl-glutaminyl–glycinyl-methoxypolyethylene glycol (CBZ-QG-mPEG) was prepared by introducing carboxybenzyl-glutaminyl-glycine (CBZ-QG) to mPEG amine. The analysis by Fourier transform infrared spectroscopy and SDS-PAGE showed that CBZ-QG-mPEG was successfully synthesized and can be recognized by mTG as an acyl donor to modify therapeutic protein, cytochrome c (cyt c). Finally, under an optimized condition (cyt c 0.5 mg/ml, CBZ-QG-mPEG 11.25 mg/ml, mTG 0.5 mg/ml, 37 °C, 2 h), the PEGylation yield reached 76.5 %.

Conclusions

This is the first study regarding the PEGylation of protein at the level of Lys residues catalyzed by mTG. The novel method could be employed to immobilize active proteins and modify therapeutic proteins.

     

Overexpression of Sirtuin2 prevents high glucose-induced vascular endothelial cell injury by regulating the p53 and NF-κB signaling pathways

Objectives

To investigate the potential role and underlying mechanism of Sirtuin2 (SIRT2) in regulating high glucose (HG)-induced vascular endothelial cell injury by using human umbilical vein endothelial cells (HUVECs).

Results

SIRT2 mRNA and protein expression levels were decreased in HG-treated HUVECs. SIRT2 overexpression increased viability, decreased apoptosis and reduced levels of reactive oxygen species in HG-treated HUVECs. SIRT2 overexpression decreased TNF-α expression (146.5 ± 22.8 pg TNF-α ml^?1) relative to that in the empty vector group (263.5 ± 18.5 pg TNF-α ml^?1) and decreased MCP-1 expression (63.8 ± 9.85 pg MCP-1 ml^?1) relative to that in the empty vector group (105.8 ± 8.5 pg MCP-1 ml^?1). SIRT2 overexpression decreased the acetylation of p53 by 33% and decreased the acetylation of NF-κB p65 by 58% in HG-treated HUVECs.

Conclusion

SIRT2 prevents HG-induced vascular endothelial cell injury through suppressing the p53 and NF-κB signaling pathways.

     

Effect of day 3 embryo morphometrics and morphokinetics on survival and implantation after slow freezing-thawing and after vitrification-warming: a retrospective cohort study

Background

Morphometric and morphokinetic evaluation of in vitro cultured human embryos allows evaluation without time restriction and reduces intra- and inter-observer variability. Even though these technologies have been reported to improve the quality of cleavage stage embryo evaluation during fresh culture, possible advantages in the evaluation of cryopreserved embryos have been scarcely explored. This study aims to compare morphometric and morphokinetic parameters between slow frozen and vitrified embryos and to determine their relationship to embryo survival and implantation rate (IR) after thawing/warming.

Methods

During fresh culture, morphometric characteristics (Total Cell Volume (TCV), symmetry, fragmentation and number of blastomeres) were measured in 286 thawed/warmed embryos. Likewise, after thawing/warming, similar morphometric characteristics were measured in 135 survived embryos. Moreover, morphokinetic parameters (time to mitosis resumption and time to compaction) were measured in 90 embryos after thawing/warming. Then, using linear regression, we investigated the differences between vitrified and slow frozen embryos and the relation of the measured characteristics to embryo survival and IR. Statistical corrections were applied to account for data clustering and for multiple testing.

Results

Vitrified embryos resume mitosis and start compaction significantly earlier than slow frozen embryos. Mitosis resumption rate was 82% for vitrified and 63% for slow frozen embryos and median time to mitosis resumption was 7.6 h and 13.1 h (p = 0.02), respectively. Compaction rate was 62% in vitrified and only 23% in slow frozen embryos. Median time to compaction was 18.1 h for vitrified embryos but, for slow frozen could not be computed since less than half of the slow frozen embryos reached compaction (p = 0.0001). Moreover, intact embryos resume mitosis significantly earlier than not intact ones regardless of the freezing method (rate: 79% vs. 66%, median time: 7.6 h vs 14.6 h, respectively, p = 0.03). Regarding morphometrics, slow frozen embryos showed lower TCV and higher blastomere symmetry after thawing than vitrified embryos despite having similar blastomere number. IR was related to blastomere number at cryopreservation in slow frozen embryos, but not in vitrified ones.

Conclusions

Interestingly, vitrified/warmed embryos undergo mitosis resumption and compaction significantly earlier than slow frozen/thawed embryos. However, the clinical use of this morphokinetic parameters still remains to be investigated in larger studies.

Trial registration

Retrospectively registered on December 15, 2015 NCT02639715.

     

Dexamethasone downregulates SIRT1 and IL6 and upregulates EDN1 genes in stem cells derived from gingivae via the AGE/RAGE pathway

Objectives

To evaluate the effects of dexamethasone on the aging of mesenchymal stem cells from human gingiva using next-generation sequencing.

Results

Four mRNAs were upregulated and 12 were downregulated when the results of dexamethasone at 24 h were compared with the control at 24 h. Expressions of SIRT1 and IL6 were decreased in dexamethasone at 24 h but expression of EDN1 was increased.

Conclusions

Application of dexamethasone reduced the expression of SIRT1 and IL6 but enhanced the expression of EDN1 of stem cells.

     

Does centrifugation matter? Centrifugal force and spinning time alter the plasma metabolome

Background

Centrifugation is an indispensable procedure for plasma sample preparation, but applied conditions can vary between labs.

Aim

Determine whether routinely used plasma centrifugation protocols (1500×g 10 min; 3000×g 5 min) influence non-targeted metabolomic analyses.

Methods

Nuclear magnetic resonance spectroscopy (NMR) and High Resolution Mass Spectrometry (HRMS) data were evaluated with sparse partial least squares discriminant analyses and compared with cell count measurements.

Results

Besides significant differences in platelet count, we identified substantial alterations in NMR and HRMS data related to the different centrifugation protocols.

Conclusion

Already minor differences in plasma centrifugation can significantly influence metabolomic patterns and potentially bias metabolomics studies.

     

R-wave synchronised atrial pacing in pediatric patients with postoperative junctional ectopic tachycardia: the atrioventricular interval investigated by computational analysis and clinical evaluation

Background

R-wave synchronised atrial pacing is an effective temporary pacing therapy in infants with postoperative junctional ectopic tachycardia. In the technique currently used, adverse short or long intervals between atrial pacing and ventricular sensing (AP–VS) may be observed during routine clinical practice.

Objectives

The aim of the study was to analyse outcomes of R-wave synchronised atrial pacing and the relationship between maximum tracking rates and AP–VS intervals.

Methods

Calculated AP–VS intervals were compared with those predicted by experienced pediatric cardiologist.

Results

A maximum tracking rate (MTR) set 10 bpm higher than the heart rate (HR) may result in undesirable short AP–VS intervals (minimum 83 ms). A MTR set 20 bpm above the HR is the hemodynamically better choice (minimum 96 ms). Effects of either setting on the AP–VS interval could not be predicted by experienced observers. In our newly proposed technique the AP–VS interval approaches 95 ms for HR > 210 bpm and 130 ms for HR < 130 bpm. The progression is linear and decreases strictly (? 0.4 ms/bpm) between the two extreme levels.

Conclusions

Adjusting the AP–VS interval in the currently used technique is complex and may imply unfavorable pacemaker settings. A new pacemaker design is advisable to allow direct control of the AP–VS interval.

     

Response of Degarelix treatment in human prostate cancer monitored by HR-MAS <Superscript>1</Superscript>H NMR spectroscopy

Introduction

The androgen receptor (AR) is the master regulator of prostate cancer cell metabolism. Degarelix is a novel gonadotrophin-releasing hormone blocker, used to decrease serum androgen levels in order to treat advanced human prostate cancer. Little is known of the rapid metabolic response of the human prostate cancer tissue samples to the decreased androgen levels.

Objectives

To investigate the metabolic responses in benign and cancerous tissue samples from patients after treatment with Degarelix by using HRMAS ¹H NMR spectroscopy.

Methods

Using non-destructive HR-MAS ¹H NMR spectroscopy we analysed the metabolic changes induced by decreased AR signalling in human prostate cancer tissue samples. Absolute concentrations of the metabolites alanine, lactate, glutamine, glutamate, citrate, choline compounds [t-choline = choline + phosphocholine (PC) + glycerophosphocholine (GPC)], creatine compounds [t-creatine = creatine (Cr) + phosphocreatine (PCr)], taurine, myo-inositol and polyamines were measured in benign prostate tissue samples (n = 10), in prostate cancer specimens from untreated patients (n = 7) and prostate cancer specimens from patients treated with Degarelix (n = 6).

Results

Lactate, alanine and t-choline concentrations were significantly elevated in high-grade prostate cancer samples when compared to benign samples in untreated patients. Decreased androgen levels resulted in significant decreases of lactate and t-choline concentrations in human prostate cancer biopsies.

Conclusions

The reduced concentrations of lactate and t-choline metabolites due to Degarelix could in principle be monitored by in vivo ¹H MRS, which suggests that it would be possible to monitor the effects of physical or chemical castration in patients by that non-invasive method.

     

Mid- and long-term correlations of plasma metabolite concentrations measured by a targeted metabolomics approach

Introduction

Plasma metabolites measured by metabolomics techniques have been shown to be associated with chronic disease risk in epidemiological studies. However, in most prospective studies metabolomic profiles can only be obtained at a single time point and data on intra-individual variation in metabolite levels over time are sparse.

Objectives

Here, we evaluated the intra-individual variation in the concentrations of 177 metabolites over time using repeat blood samples of 104 adults (50% female).

Methods

Blood samples were obtained at a baseline visit between 1994 and 1998 and during two further examinations 14 and 15 years later. Plasma metabolite levels were quantified by tandem mass spectrometry with the MetaDisIDQ? p180 Kit. Intra-individual variation was assessed by Spearman’s correlation coefficients (ρ).

Results

Mid-term correlations over 1 year were good (ρ ≥ 0.7) for 5.1% and reasonable (ρ ≥ 0.4 < 0.7) for 61.0% of the metabolites, while long-term correlations over 15 years were good for 2.8% and reasonable for 27.1%. The strongest mid-term correlations between metabolite concentrations were observed for the acylcarnitine C₃–OH (0.72) and metabolites from the amino acid/biogenic amine groups, i.e. creatinine (0.83), proline (0.79), lysine (0.77), isoleucine (0.76), and ornithine (0.74). C₃–OH (0.78) as well as several amino acids/biogenic amines, i.e. ornithine (0.76), sarcosine (0.76), lysine (0.75), spermine (0.73), and glutamine (0.69) showed the strongest long-term correlations.

Conclusions

The biological reproducibility of plasma concentrations of a majority of metabolites occurs reasonable over 1 year. In contrast, concentrations of many metabolites seem to be affected by substantial intra-individual variation over 15 years.

     

Dichorionic triplets following frozen-thawed poor-stage embryo transfer: a report of two cases and a review

Background

We describe two cases of dichorionic triplet pregnancy after a frozen-thawed poor-stage embryo transfer.

Main body of the abstract

A 39-year-old and a 41-year-old woman underwent ART treatment. The first patient underwent intracytoplasmic sperm injection (ICSI) at 34 years of age, and two frozen-thawed poor-stage embryos were transferred at 39 years of age with assisted hatching, resulting in a trichorionic triamniotic triplet pregnancy. The second patient underwent ICSI, and two poor-grade blastocysts were transferred followed by assisted hatching, resulting in a dichorionic triamniotic triplet pregnancy.In the first case, the heartbeat of one monozygotic twin fetus had stopped on day 48 post-transfer (9 weeks 2 days), resulting in a dichorionic diamniotic twin pregnancy. A healthy boy and girl were delivered by elective caesarean section at 36 weeks, 5-days gestation. In the second case, the patient underwent selective reduction of the monochorionic twins, resulting in a single pregnancy that was vaginally delivered without any problems at 38 weeks 0-days gestation.

Short conclusions

Numerous factors may be associated with the development of a monochorionic pregnancy; however, controversies still remain. The present morphological grading for embryos is insufficient for inhibiting the development of a monochorionic pregnancy.

     

Transient expression of recombinant tissue plasminogen activator (rt-PA) gene in cucurbit plants using viral vector

Objective

To use a transient expression system to express a truncated human tissue plasminogen activator (K2S) gene in cucurbit plants.

Results

The recombinant tissue plasminogen activator protein (K2S form) was expressed in active form in cucurbit plants. Its molecular weight was 43 kDa. The plant-derived rt-PA was determined using goat anti-rabbit antibody by western blotting. Among the infected lines, the highest expression of rt-PA was 62 ng/100 mg per leaf tissue as measured by ELISA. The enzymatic activity of the plant-derived rt-PA was 0.8 IU/ml.

Conclusions

The K25 form of rt-PA was expressed for the first time using the viral expression system. Plant-derived rt-PA showed similar potency to commercially-available PA.