PCI-24781 can improve in vitro and in vivo developmental capacity of pig somatic cell nuclear transfer embryos

Objective

To examine the effect of PCI-24781 (abexinostat) on the blastocyst formation rate in pig somatic cell nuclear transferred (SCNT) embryos and acetylation levels of the histone H3 lysine 9 and histone H4 lysine 12.

Results

Treatment with 0.5 nM PCI-24781 for 6 h significantly improved the development of cloned embryos, in comparison to the control group (25.3 vs. 10.5 %, P < 0.05). Furthermore, PCI-24781 treatment led to elevated acetylation of H3K9 and H4K12. TUNEL assay and Hoechst 33342 staining revealed that the percentage of apoptotic cells in blastocysts was significantly lower in PCI-24781-treated SCNT embryos than in untreated embryos. Also, PCI-24781-treated embryos were transferred into three surrogate sows, one of whom became pregnant and two fetuses developed.

Conclusion

PCI-24781 improves nuclear reprogramming and the developmental potential of pig SCNT embryos.

     

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.

     

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.

     

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.

     

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.

     

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.

     

Impact of post-collection freezing delay on the reliability of serum metabolomics in samples reflecting the California mid-term pregnancy biobank

Background

Population-based biorepositories are important resources, but sample handling can affect data quality.

Objective

Identify metabolites of value for clinical investigations despite extended postcollection freezing delays, using protocols representing a California mid-term pregnancy biobank.

Methods

Blood collected from non-pregnant healthy female volunteers (n?=?20) underwent three handling protocols after 30 min clotting at room temperature: (1) ideal—samples frozen (??80 °C) within 2 h of collection; (2) delayed freezing—samples held at room temperature for 3 days, then 4 °C for 9 days, the median times for biobank samples, and then frozen; (3) delayed freezing with freeze–thaw—the delayed freezing protocol with a freeze–thaw cycle simulating retrieved sample sub-aliquoting. Mass spectrometry-based untargeted metabolomic analyses of primary metabolism and complex lipids and targeted profiling of oxylipins, endocannabinoids, ceramides/sphingoid-bases, and bile acids were performed. Metabolite concentrations and intraclass correlation coefficients (ICC) were compared, with the ideal protocol as the reference.

Results

Sixty-two percent of 428 identified compounds had good to excellent ICCs, a metric of concordance between measurements of samples handled with the different protocols. Sphingomyelins, phosphatidylcholines, cholesteryl esters, triacylglycerols, bile acids and fatty acid diols were the least affected by non-ideal handling, while sugars, organic acids, amino acids, monoacylglycerols, lysophospholipids, N-acylethanolamides, polyunsaturated fatty acids, and numerous oxylipins were altered by delayed freezing. Freeze–thaw effects were assay-specific with lipids being most stable.

Conclusions

Despite extended post-collection freezing delays characteristic of some biobanks of opportunistically collected clinical samples, numerous metabolomic compounds had both stable levels and good concordance.

     

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.

     

Thaw pond development and initial vegetation succession in experimental plots at a Siberian lowland tundra site

Background and aims

Permafrost degradation has the potential to change the Arctic tundra landscape. We observed rapid local thawing of ice-rich permafrost resulting in thaw pond formation, which was triggered by removal of the shrub cover in a field experiment. This study aimed to examine the rate of permafrost thaw and the initial vegetation succession after the permafrost collapse.

Methods

In the experiment, we measured changes in soil thaw depth, plant species cover and soil subsidence over nine years (2007–2015).

Results

After abrupt initial thaw, soil subsidence in the removal plots continued indicating further thawing of permafrost albeit at a much slower pace: 1 cm y^?1 over 2012–2015 vs. 5 cm y^?1 over 2007–2012. Grass cover strongly increased after the initial shrub removal, but later declined with ponding of water in the subsiding removal plots. Sedges established and expanded in the wetter removal plots. Thereby, the removal plots have become increasingly similar to nearby ‘natural’ thaw ponds.

Conclusions

The nine years of field observations in a unique shrub removal experiment at a Siberian tundra site document possible trajectories of small-scale permafrost collapse and the initial stage of vegetation recovery, which is essential knowledge for assessing future tundra landscape changes.

     

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.

     

Evaluation of cervical posture improvement of children with cerebral palsy after physical therapy based on head movements and serious games

Background

This paper presents the preliminary results of a novel rehabilitation therapy for cervical and trunk control of children with cerebral palsy (CP) based on serious videogames and physical exercise.

Materials

The therapy is based on the use of the ENLAZA Interface, a head mouse based on inertial technology that will be used to control a set of serious videogames with movements of the head.

Methods

Ten users with CP participated in the study. Whereas the control group (n = 5) followed traditional therapies, the experimental group (n = 5) complemented these therapies with a series of ten sessions of gaming with ENLAZA to exercise cervical flexion–extensions, rotations and inclinations in a controlled, engaging environment.

Results

The ten work sessions yielded improvements in head and trunk control that were higher in the experimental group for Visual Analogue Scale, Goal Attainment Scaling and Trunk Control Measurement Scale (TCMS). Significant differences (27% vs. 2% of percentage improvement) were found between the experimental and control groups for TCMS (p < 0.05). The kinematic assessment shows that there were some improvements in the active and the passive range of motion. However, no significant differences were found pre- and post-intervention.

Conclusions

Physical therapy that combines serious games with traditional rehabilitation could allow children with CP to achieve larger function improvements in the trunk and cervical regions. However, given the limited scope of this trial (n = 10) additional studies are needed to corroborate this hypothesis.

     

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.

     

Sperm quality and paternal age: effect on blastocyst formation and pregnancy rates

Background

Several studies suggest a decrease in sperm quality in men in the last decades. Therefore, the aim of this work was to assess the influence of male factors (sperm quality and paternal age) on the outcomes of conventional in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI).

Methods

This retrospective study included all couples who underwent IVF or ICSI at Montpellier University Hospital, France, between 1 January 2010 and 31 December 2015. Exclusion criteria were cycles using surgically retrieved sperm or frozen sperm, with pre-implantation genetic diagnosis or using frozen oocytes. The primary outcomes were the blastulation rate (number of blastocysts obtained at day 5 or day 6/number of embryos in prolonged culture at day 3) and the clinical pregnancy rate. The secondary outcomes were the fertilization and early miscarriage rates.

Results

In total, 859 IVF and 1632 ICSI cycles were included in this study. The fertilization rate after ICSI was affected by oligospermia. Moreover, in ICSI, severe oligospermia (lower than 0.2 million/ml) led to a reduction of the blastulation rate. Reduced rapid progressive motility affected particularly IVF, with a decrease of the fertilization rate and number of embryos at day 2 when progressive motility was lower than 32%.Paternal age also had a negative effect. Although it was difficult to eliminate the bias linked to the woman’s age, pregnancy rate was reduced in IVF and ICSI when the father was older than 51 and the mother older than 37 years.

Conclusions

These results allow adjusting our strategies of fertilization technique and embryo transfer. In the case of severe oligospermia, transfer should be carried out at the cleaved embryo stage (day 2–3) due to the very low blastulation rate. When the man is older than 51 years, couples should be aware of the reduced success rate, especially if the woman is older than 37 years. Finally, promising research avenues should be explored, such as the quantification of free sperm DNA, to optimize the selection of male gametes.

     

Perioperative Duloxetine and Etoricoxibto improve postoperative pain after lumbar Laminectomy: a randomized,double-blind,controlled study

Background

Duloxetine, Etoricoxib and opioid are of the commonly administered drugs in Lumbar laminectomy. The aim of this study is to assess the effect of perioperative use of Duloxetine in combination with Etoricoxib on postoperative pain and opioid requirements.

Methods

One hundred twenty patients with ASA physical status were enrolled with age between 18 and 70 years. Patients were divided randomly into four groups of 30 patients: group P received placebo, group E received etoricoxib 120 mg, group D received duloxetine 60 mg and group D/E received duloxetine 60 mg capsules and etoricoxib 120 mg; 1 h before surgery and 24 h after.

Results

Neither Duloxetine nor etoricoxib individually had effect on pain with movement, while their combination revealed a significant reduction in pain scores over the entire postoperative period at rest and on movement. Etoricoxib showed a significant decrease in pain at all times at rest when compared with group P, while it showed significant pain decrease only at 0, 2 and 4 h when compared with group D. On the other hand duloxetine alone showed significant decrease in pain at rest at 24 h and 48 h when compared with group P. ConcerningMorphine requirement after 24 h.; it wassignificantly lower in the D/E group in comparison with groups P, E and D. It should be noted also that there was a significant decrease morphine requirement in both groups E and D.

Conclusion

The perioperative administration of the combination of etoricoxib and duloxetine improved analgesia and reduced opioid consumption without significant side effects.

Trial registration

ISRCTN48329522. 17 June 2017

     

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.

     

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.

     

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.

     

New insights into the mechanistic action of methyldehydrodieugenol B towards <Emphasis Type="Italic">Leishmania</Emphasis> (L.) <Emphasis Type="Italic">infantum</Emphasis> via a multiplatform based untargeted metabolomics approach

Introduction

Leishmaniasis is a parasitic neglected disease affecting millions of people worldwide. Clinical practice resorts to long and costly treatments with a therapeutic arsenal limited to highly toxic drugs, often associated to adverse side effects. Additionally, resistant strains are reported to be increasing.

Aim

In this work, the mechanistic action of a drug candidate (methydehydrodieugenol B), isolated from twigs of Nectandra leucantha, towards Leishmania infantum was studied by a global metabolomics approach using GC-MS and RPLC-MS platforms.

Method

L. infantum promastigotes were grown in culture medium for 72 h and treated with methydehydrodieugenol B at 58.18 μg.mL-1 concentration; after 48 h treatment, enzyme activity was quenched, cells washed and frozen until analysis. For GC-MS analysis (Fiehn’s method), 1:1 methanol:water extracts were prepared and derivatized with O-methoxyamine in pyridine at room temperature for 90 min, followed by silylation with BSTFA/1% TMCS at 40 °C for 30 min. Pure methanolic extracts were also prepared and analyzed directly by RPLC-MS with a acetonitrile/water mobile phase acidulated with formic acid and gradient elution.

Result

Several amino acids, fatty acids, carbohydrates, and glycerolipids were found as discriminant metabolites, mostly decreased in treated samples. Due to the complexity of the parasite metabolism and the great diversity of altered metabolites, a multi-target mechanism was assigned to the drug candidate, where changes in the cell energy sources and in the lipid composition of the parasite plasma membrane were prominent.

Conclusion

These results contributed to elucidate the broad action of methyldehydrodieugenol B against Leishmania, paving the way in the search of novel alternative therapies.

     

Daily variation and effect of dietary folate on urinary pteridines

Introduction

Urinary pteridines are putative molecular biomarkers for noninvasive cancer screening and prognostication. Central to their translational biomarker development is the need to understand the sources and extent of their non-epidemiological variation.

Objectives

This study was designed to characterize the two primary sources of urinary pteridine variance: daily variation and the effect of dietary folate.

Methods

Daily variation was studied by collecting urine specimens (n = 81) three times daily for 3 days. The effect of dietary folate was investigated in a treatment study in which urine specimens (n = 168) were collected daily during a control week and a treatment week during which participants received dietary folate supplements. Measurements of six urinary pteridines were made using high-performance liquid chromatography–tandem mass spectrometry. Coefficients of variation were calculated to characterize daily variance between and within subjects, while nearest neighbor non-parametric analyses were used to identify diurnal patterns and measure dietary folate effects.

Results

Daily variance was approximately 35 % RSD for both within-day and between-day periods for most pteridines. Diurnal patterns in response to circadian rhythms were similarly observed for urinary pteridines. Folate supplementation was shown to alter urinary pteridine profiles in a pathway dependent manner, suggesting that dietary folate may regulate endogenous neopterin and biopterin biosynthesis.

Conclusions

Urinary pteridine levels were found to be responsive to both daily variation and folate supplementation. These findings provide new insights into pteridine biosynthesis and regulation as well as useful information for the design of future clinical translational research.

     

Effect of food intake on myocardial performance index

Background

Myocardial performance index (MPI) has been investigated in a variety of populations, but the effect of food intake has not been evaluated. We assessed whether myocardial performance index is affected by food intake in healthy subjects.

Methods

Twenty-three healthy subjects aged 25.6?±?4.5 years were investigated. MPI was measured before, 30 min after, and 110 min after a standardized meal.

Results

MPI decreased significantly (P?<?0.05) from fasting values 30 min after the meal, and had almost returned to baseline after 110 min. MPI decreased from 0.28?±?0.06 (fasting) to 0.20?±?0.07 30 min after eating. At 110 min after eating the index value was almost back to the baseline value 0.26?±?0.06. (P?=?0.15).

Conclusions

This study shows that myocardial performance index is affected by food intake in healthy subjects.