High Fragmentation Rates Research Articles

Regard typo-technologique sur les productions lithiques foliacées du Solutréen de la vallée de l’Erve (Mayenne, France)

Regard typo-technologique sur les productions lithiques foliacées du Solutréen de la vallée de l’Erve (Mayenne, France)

Shock wave lithotripsy outcomes for lower pole and non-lower pole stones from a university teaching hospital: Parallel group comparison during the same time period.

Introduction:Shock wave lithotripsy (SWL) is a treatment option for all locations of renal and ureteric stones. We compared the results of SWL for lower pole renal stones with all other non-lower pole renal and ureteric stones during the same time period.Material and Methods:All SWL procedures were carried out as day case procedures by a mobile lithotripter from January 2012 to August 2013. The follow-up imaging was a combination of KUB X-ray or USS. Following SWL treatment, the stone free rate (SFR) was defined as ≤3 mm fragments.Results:A total of 148 patients with a mean age of 62 years underwent 201 procedures. Of the 201 procedures, 93 (46%) were for lower pole stones. The non-lower pole stones included upper pole (n = 36), mid pole (n = 40), renal pelvis (n = 10), PUJ (n = 8), mid ureter (n = 3), upper ureter (n = 5) and a combination of upper, middle and/or lower pole (n = 6). The mean stone size for lower pole stones (7.4 mm; range: 4-16 mm) was slightly smaller than non-lower pole stones (8 mm; range: 4-17 mm). The stone fragmentation was successful in 124 (62%) of patients. However, the SFR was statistically significantly better (P = 0.023) for non-lower pole stones 43 (40%) compared to lower pole stones 23 (25%). There were 9 (4%) minor complications and this was not significantly different in the two groups.Conclusions:Although SWL achieves a moderately high stone fragmentation rate with a low complication rate, the SFR is variable depending on the location of stone and the definition of SFR, with lower pole stones fairing significantly worse than stones in all other locations.

Cell-free DNA in human follicular fluid as a biomarker of embryo quality

Could cell-free DNA (cfDNA) quantification in individual human follicular fluid (FF) samples become a new non-invasive predictive biomarker for in vitro fertilization (IVF) outcomes? CfDNA level in human follicular fluid samples was significantly correlated with embryo quality and could be used as an innovative non-invasive biomarker to improve IVF outcomes. CfDNA fragments, resulting from apoptotic or necrotic events, are present in the bloodstream and their quantification is already used as a biomarker for gynaecological and pregnancy disorders. Follicular fluid is important for oocyte development and contains plasma components and factors secreted by granulosa cells during folliculogenesis. CfDNA presence in follicular fluid and its potential use as an IVF outcome biomarker have never been investigated. One hundred individual follicular fluid samples were collected from 43 female patients undergoing conventional IVF (n = 26) or ICSI (n = 17). CfDNA level was quantified in each individual follicular fluid sample. At oocyte collection day, follicles were aspirated individually. Only blood-free follicular fluid samples were included in the study. Follicle size was calculated based on the follicular fluid volume. Each corresponding cumulus-oocyte complex was isolated for IVF or ICSI procedures. Follicular fluid cfDNA was measured by quantitative PCR with ALU-specific primers. Human follicular fluid samples from individual follicles contain measurable amounts of cfDNA (mean ± SD, 1.62 ± 2.08 ng/µl). CfDNA level was significantly higher in small follicles (8-12 mm in diameter) than in large ones (>18 mm) (mean ± SD, 2.54 ± 0.78 ng/µl versus 0.71 ± 0.44 ng/µl, respectively, P = 0.007). Moreover, cfDNA concentration was significantly and negatively correlated with follicle size (r = -0.34; P = 0.003). A weak significant negative correlation between DNA integrity and 17β-estradiol level in follicular fluid samples at oocyte collection day was observed (r = -0.26; P = 0.008). CfDNA level in follicular fluid samples corresponding to top quality embryos was significantly lower than in follicular fluid samples related to poor quality embryos (P = 0.022). Similarly, cfDNA level was also significantly lower in follicular fluid samples related to embryos with low fragmentation rate (≤25%) than with high fragmentation rate (>25%) (P = 0.02). A larger study should be conducted in order to establish the predictive value of cfDNA level for embryo quality and to investigate whether follicular fluid cfDNA levels are correlated with embryo implantation rates and pregnancy outcomes. Moreover, the role of follicular fluid cfDNA on embryo quality should be studied to determine whether high cfDNA concentration in follicular fluid is only a consequence or also a cause of follicular dysfunction. CfDNA evaluation in individual follicular fluid samples might represent an innovative biomarker of embryo quality to use as a supplemental tool to predict embryo quality during IVF. This study was partially supported by the University Hospital of Montpellier and Ferring Pharmaceuticals. The authors of the study have no competing interests to report.

Ecology of Aquatic WarblersAcrocephalus paludicolain a Fall Stopover Area on the Atlantic Coast of France

The Aquatic Warbler Acrocephalus paludicola is the only endangered songbird in continental Europe. This trans-Saharan migratory bird significantly transits along the French Atlantic coastline during post-breeding migration and the right bank of the Gironde estuary has been identified as an important stopover site. We studied the spatial occupancy strategies of stationing individuals during August of three successive years (2010–2012). We characterized habitat use by radio-tracking individuals revealing relatively small foraging ranges (6.6 ± 2.6 ha on average) with only 1 ha actually exploited (core area), and a relatively high habitat fragmentation rate. Capture-mark-recapture analyses assessed the average stopover duration of individuals and body-mass variations during their stopover. The estimated average stopover duration was 6.46 ± 0.46 days (95% confidence interval: 4.4–9.6). Lean birds tended to forage significantly more than stout birds: on average, they gain 2.81 ± 0.89% of their initial mass each day whereas stout birds only gain 0.12 ± 0.56%. Analyses of droppings characterized the local diet. We noticed that Aquatic Warbler preferentially used partially-flooded or flooded habitats with heterogeneous and rather low vegetation, such as bulrush beds or bulrush beds mixed with reed beds. Orthoptera, Araneae and Hymenoptera represented the largest contributions to the consumed biomass (64.7%, 13.4% and 8.9% respectively). The importance of the fuel deposition rate of lean birds reflects the importance of the estuary as a stopover site for the species. It means that the available resources allow birds to replenish and continue their migration route. However, the sustainability of the site's functionality is questioned because of the evolution of habitats (erosion, rise in water levels and changes in food web).

High Intrinsic Mechanical Flexibility of Mouse Prion Nanofibrils Revealed by Measurements of Axial and Radial Young’s Moduli

Self-templated protein aggregation and intracerebral deposition of aggregates, sometimes in the form of amyloid fibrils, is a hallmark of mammalian prion diseases. What distinguishes amyloid fibrils formed by prions from those formed by other proteins is not clear. On the basis of previous studies on yeast prions that correlated high intrinsic fragmentation rates of fibrils with prion propagation efficiency, it has been hypothesized that the nanomechanical properties of prion amyloid such as strength and elastic modulus may be the distinguishing feature. Here, we reveal that fibrils formed by mammalian prions are relatively soft and clearly in a different class of rigidities when compared to nanofibrils formed by nonprions. We found that amyloid fibrils made of both wild-type and mutant mouse recombinant PrP(23-231) have remarkably low axial elastic moduli of 0.1-1.4 GPa. We demonstrate that even the proteinase K resistant core of these fibrils has similarly low intrinsic rigidities. Using a new mode of atomic force microscopy called AM-FM mode, we estimated the radial modulus of PrP fibrils at ∼0.6 GPa, consistent with the axial moduli derived by using an ensemble method. Our results have far-reaching implications for the understanding of protein-based infectivity and the design of amyloid biomaterials.

Habitat fragmentation and the genetic structure of the Amazonian palm Mauritia flexuosa L.f. (Arecaceae) on the island of Trinidad

Studies of small-scale spatial genetic structure are crucial to the implementation of successful conservation and management plans as they enhance our capacity to predict a population’s ability to respond to changing environments. This is especially true in the tropics, where high rates of fragmentation and land-use change create potential barriers to genetic exchanges. We examined the genetic diversity of wild populations of the economically important palm, Mauritia flexuosa (Arecaceae) in and around the Aripo Savannas Scientific Reserve on the island of Trinidad. In a small collection area of roughly 10 km2 we found both spatial and inter-generational, or temporal, genetic differentiation and high levels of nearest-neighbor mating. We suggest that these spatial and temporal genetic patterns reflect recent and drastic anthropogenic changes to the landscape of the study sites, which we hypothesize have created barriers to gene flow through animal-mediated seed dispersal and pollination.

Tracking Acropora fragmentation and population structure through thermal-stress events

Fragmentation of reef corals spreads the risk of encountering unfavorable environments. High rates of fragmentation, however, may give the false impression of expanding, diversifying populations when instead the populations are simply cloning. We developed a discrete model that tracked the contribution of fragments to the coral populations by monitoring two thermally sensitive corals, branching coral Acropora palmata and corymbose Acropora colonies before, during, and after thermal-stress events at Haulover Bay, in the US Virgin Islands (Caribbean) and at Aka Jima in southern Japan (Pacific), respectively. We hypothesized that fragmentation rates would increase following disturbance years for both Acropora taxa. Using a Bayesian approach we tested the fit of the models with the observed data. Fragmentation was more frequent for A. palmata in the Caribbean than for corymbose Acropora in southern Japan. The highest frequency of fragmentation for A. palmata occurred during and soon after thermal stress, increasing the population density but shifting the size-frequency distribution toward small colonies. Fragmentation of corymbose Acropora in southern Japan was generally low and relatively constant throughout the study, except during thermal stress, which resulted in the loss of the smallest and largest colonies, effectively narrowing the size-frequency distribution.

2302 A ROLE FOR HEDGEHOG SIGNALLING IN URETERAL STENT INDUCED MORBIDITY

INTRODUCTION AND OBJECTIVES: To assess the fragmentation efficiency of laser lithotripsy along a wide range of pulse energies, frequencies, power settings and different laser fibers, paying special attention to compare high frequency to low frequency lithotripsy using a dynamic and innovative testing procedure free from any human interaction bias. METHODS: An automated, in saline submerged laser fragmentation testing system was developed, where laser emission took place during a pre-determined period of time (30s) with laser fibers moving at constant velocity over the surface of artificial stones (plaster of Paris) and creating a fissure, whose wide, depth and volume were analyzed. Lithotripter settings included different pulse energies (0.2-1.2J), frequencies (5-40Hz), power levels (4-20W) and laser fiber diameters (200 and 550 m), including high frequency vs. low frequency experiments maintaining the same total power, totaling 34 different experimental conditions. RESULTS: Low frequency with high pulse energy settings showed to be several times (2.1-6.2x) more efficient than high frequency with low pulse energy at the same power level for all levels tested (p 0.00001) and was also associated with deeper (p 0.01) and wider (p 0.00001) fissures. A linear correlation was found between pulse energy and fragmentation volume (p 0.00001), fissure wide (p 0.00001) and fissure depth (p 0.00001). No correlation was found between power (W) used and fragmentation volume (p 0.29), fissure depth (p 0.06) or fissure wide (p 0.7). Large fibers are associated with wider fissures (p 0.00001), small fibers with deeper fissures (p 0.00001), whatever the frequency, pulse energy or total power used. No differences were detected concerning laser fiber diameter and fragmentation volume (p 0.81) except at very low pulse energies (0.2J) were the large fiber was less efficient (p 0.015). CONCLUSIONS: At the same total power level, low frequency laser lithotripsy is the most efficient laser lithotripter setting. Elevated power settings using high frequency do not warrant higher fragmentation rates when compared to low frequency high pulse energy settings, even those of several orders of magnitude below. These results emphasize that pulse energy seems to be the key feature in fragmentation efficacy, downplaying both the role of high-frequency and total power setting. Source of Funding: None

Fish of the Taiassuí and Benfica river basins, Benevides, Pará (Brazil)

Species inventories are relatively efficient and inexpensive tools for the monitoring of biodiversity, especially in areas that have suffered high rates of deforestation, such as the municipalities located within the “Belém Endemism Center”. This region extends from northeastern Pará to northern Maranhão and has a unique flora and fauna, but is threatened by high rates of deforestation and habitat fragmentation since it is included in the so-called “Arc of Deforestation”. Benevides is the third most impacted municipality within the metropolitan region of Belém (Pará), after Belém itself and Santa Bárbara, but the biological diversity of its freshwater fish fauna is still unknown. In the present study, 81 fish species belonging to seven orders and 23 families were identified. Characidae and Cichlidae were the families with the largest numbers of species in the Taiassuí and Benfica river basins. Two of the species – Apistogramma tucurui and Hyphessobrycon inconstans – were recorded in the state for the first time. Based on the composition analysis of the fishes, it is possible to said that the Benevides fish community is relatively well preserved, because it has high diversity of groups.

The Incidence of Aneuploidy Significantly Increases After the Age of 40 in Women With a History of Recurrent Pregnancy Loss

To determined if patients undergoing in vitro fertilization (IVF) and preimplantation genetic screening (PGS) for recurrent pregnancy loss (RPL) experienced increased rates of aneuploidy with increasing maternal age. A retrospective review was conducted of all embryos that underwent PGS by 23 chromosome SNP microarrays from January 2010 to January 2011.Patients underwent standard IVF and PGS primarily due to a history of ≥ 2 spontaneous miscarriages. Embryo biopsy was performed at either the cleavage or blastocyst stage. Sample DNA was amplified and analyzed using HumanCytoSNP-12 DNA beadchips and GenomeStudio and KaryoStudio software. Embryos derived from parents with known translocations or inversions were excluded from the study. Data was split into four patient groups based on maternal age (<35, 35-37, 38-40, and >40). Binomial confidence intervals for proportions were calculated. 1,903 embryos from 236 clinical IVF cycles were tested. Of embryos from women <35 years (n=594), 288 (48%) were aneuploid. Of embryos from women 35-37 years (n=268), 132 (49%) were aneuploid. Of embryos from women 38-40 years (n=626), 298 (48%) were aneuploid. Of embryos from women >40 years (n=414), 236 (57%) were aneuploid. No differences were observed among the rates of aneuploidy in the <35, 35-37, and 38-40 groups. The >40 group had an average rate of aneuploidy of 57%, significantly (P=0.001) higher than the pooled results of the <35, 35-37, and 38-40 groups. There was a high rate of morphologic fragmentation on some embryos which led to an approximate 9% of embryos with no molecular diagnosis obtained and this did not statistically differ between groups. Pregnancy loss rates increase steadily with advancing maternal age in the general population. Our data suggests that the underlying mechanisms that cause RPL in women under the age of 40 may overshadow the gradual increasing rate of aneuploidy that accompanies increasing maternal age seen in the general population. After the maternal age of 40, age related aneuploidy begins to represent an additional pregnancy challenge for couples suffering from RPL.

Deletions and duplications identified by 23 chromosome single nucleotide polymorphism (SNP) microarray are associated with aneuploidy

Deletions and duplications identified by 23 chromosome single nucleotide polymorphism (SNP) microarray are associated with aneuploidy

HBO1 Is Required for H3K14 Acetylation and Normal Transcriptional Activity during Embryonic Development

We report here that the MYST histone acetyltransferase HBO1 (histone acetyltransferase bound to ORC; MYST2/KAT7) is essential for postgastrulation mammalian development. Lack of HBO1 led to a more than 90% reduction of histone 3 lysine 14 (H3K14) acetylation, whereas no reduction of acetylation was detected at other histone residues. The decrease in H3K14 acetylation was accompanied by a decrease in expression of the majority of genes studied. However, some genes, in particular genes regulating embryonic patterning, were more severely affected than "housekeeping" genes. Development of HBO1-deficient embryos was arrested at the 10-somite stage. Blood vessels, mesenchyme, and somites were disorganized. In contrast to previous studies that reported cell cycle arrest in HBO1-depleted cultured cells, no defects in DNA replication or cell proliferation were seen in Hbo1 mutant embryo primary fibroblasts or immortalized fibroblasts. Rather, a high rate of cell death and DNA fragmentation was observed in Hbo1 mutant embryos, resulting initially in the degeneration of mesenchymal tissues and ultimately in embryonic lethality. In conclusion, the primary role of HBO1 in development is that of a transcriptional activator, which is indispensable for H3K14 acetylation and for the normal expression of essential genes regulating embryonic development.

Design of a novel Couette flow bioreactor to study the growth of fungal microorganism

Cultivations using Trichoderma reesei Rut C-30 were performed in a 5-l Couette flow bioreactor (CFB) which was designed and built to perform experiments in batch and continuous modes. Process parameters such as dissolved oxygen, pH and temperature were measured and controlled without disturbing the shear profile inside the bioreactor. Effect of shear on the growth, protein production and morphology was studied by performing runs at 100, 200, 300 and 400 rpm. At higher shear rates, lower protein production rate and activity, and higher rate of fragmentation were observed. Also, the cell thickness decreased with increasing speed, going from 8.3 μm for the experiment at 100 rpm to 4.3 μm at 400 rpm. The effect of substrate, lactose (an inducer) or glucose, was investigated by switching the feed medium during the two runs performed at 300 and 400 rpm. The novel design of the CFB used in the present study includes a large volume that allows growing larger size microorganisms (e.g. fungi) and permits larger sampling volumes without affecting the cultivation. It also has the ability to carry out experiments for long periods of time, both in batch and continuous modes.

Nesting Habitat of the Lilac‐crowned Parrot in a Modified Landscape in Mexico

ABSTRACTParrot populations are being increasingly pressured to occupy modified or fragmented landscapes, yet little is known of the habitat requirements of most species, particularly with regard to the effects on breeding habitat. We evaluated nesting habitat of the lilac‐crowned parrot Amazona finschi in the modified landscape of coastal Michoacan in Mexico. We located 90 parrot nests in 12 tree species in Michoacan, with lilac‐crowned parrots presenting a narrow niche‐breadth of tree species used for nesting. Considering an additional 82 nest trees recorded for lilac‐crowned parrots in Jalisco, we determined a 51 percent similarity in cavity resource use by parrots in the two dry forest regions. Overall, the predominant nest tree species with 76 percent of nests were Astronium graveolens, Piranhea mexicana, Brosimum alicastrum, and Tabebuia spp., all characteristic of semi‐deciduous forest. Only 8 percent of nests occurred in trees characteristic of deciduous forest. Parrots utilized large trees with canopy level cavities as nest sites, and preferred conserved semi‐deciduous forest for nesting, with fewer nests than expected in deciduous forest and transformed agricultural land. Nest areas in semi‐deciduous forest occurred on significantly steeper terrain, as remnant semi‐deciduous forest is restricted to steep ridges and canyons. Those parrot nests in modified habitats and forest patches were located near to continuous forest, with nest trees in open agricultural land being significantly closer to continuous forest than nests in disturbed forest patches. These results demonstrate the importance of conserved semi‐deciduous forest as breeding habitat for the threatened, endemic lilac‐crowned parrot, making wild populations of the species vulnerable to the high rate of transformation and fragmentation of tropical dry forest.

Efficacy and Treatment Outcome of a New Electromagnetic Lithotripter for Upper Urinary Tract Calculi

To evaluate the efficacy and treatment outcome of a new electromagnetic lithotripter-The Siemens Lithoskop-in the treatment of upper urinary tract stones. Data of 218 consecutively treated patients for upper urinary tract stones between August 2006 and December 2007 with complete follow-up was collected from a prospectively maintained data-base. All patients underwent outpatient based extracorporeal SWL with the shock wave system Pulso integrated into the Siemens Lithoskop platform. Mean surface areas of renal and ureteral stones were 65 +/- 59 mm(2) and 32 +/- 18 mm(2) respectively. Overall, 95% of urinary stones demonstrated fragmentation post SWL. After three months follow-up 84% and 88% of patients harboring renal and ureteral stones were regarded as treatment success. Patients harboring renal or ureteral stones <10 mm were successful treated in 95%. Auxiliary measures in renal and ureteral stones were necessary in 7.8 and 18.8% of patients respectively. Mean number of sessions performed for renal and ureteral stones were 1.6 +/- 1 and 1.4 +/- 0.8, respectively. No major complications as sepsis or renal hematoma were observed. Outpatient based electromagnetic SWL using the Lithoskop achieves high fragmentation and success rates with minimal analgesia requirements and a low complication rate.

The use of tandem repeat markers to detect Y chromosome mutation in infertile human spermatozoa

OBJECTIVE: Oxidative stress is a cause of male infertility, and contributes to the high rate of DNA fragmentation. Effects of oxidative damage are mediated by the highly active reactive oxygen species (ROS). ROS is generated by endogenous metabolism, inflammatory and by environmental agents. The Y chromosome is susceptible to such DNA fragmentation due to presence of repetitive elements. The instability of Y chromosome is a precursor of mutagenic changes in the germ line, leading to infertility and cancer. We hypothesize that repetitive DNA sequences in the Y chromosome in infertile patients are highly susceptible to increased mutations. DESIGN: We tested repetitive DNA sequences in the Y chromosome from infertile spermatozoa and testicular tissue to identify the loci most sensitive to oxidative stress induced mutations. MATERIALS AND METHODS: Sperm DNA from both fertile (control n=5) and infertile (n=25) were analyzed for the presence of mutations by using small-pool PCR. DNA purified from semen or testicular samples from both groups were used to detect the mutations. Moreover, DNA from matching somatic cell (blood) from these samples were analyzed to determine the size of constitutive alleles for each locus and to determine background levels of the mutations. A microsatellite marker panel was used including 12 tri- tetra- and pentanucleotide repeats on the Y chromosome(Promega). Strand breaks in spermatozoa from both groups were measured by using the sperm Chromatin Structure Assay (SCSA). RESULTS: The SP-PCR data on the infertile germ line showed a significant increased mutation frequency (P<0.05) compared to controls. No mutation was seen in matched infertile blood samples from these individuals. Likewise, none of the fertile germ line and soma samples tested displayed mutation. Our results also showed that the pentanucleotide locus on the Y chromosome is amongst the most sensitive to mutation (>30%). The most sensitive repeat motifs for germ line specific mutation are tetranucleotide repeats on the Y-chromosome which exhibited higher mutation frequency (18% to 32%). Moreover, mononucleotide repeats exhibit a wider range of sensitivity to mutation (15% to 47%). The SCSA indicate a significant increase in DNA strand breaks in the infertile group compared to the controls (P<0.05). CONCLUSIONS: Our data demonstrate the sensitivity of tandem repeat markers in the Y chromosome for detection of mutations in infertile spermatozoa. These markers may also be useful for monitoring mutagenic changes in the germ line.

Homologous Recombination Is Necessary for Normal Lymphocyte Development

Primary immunodeficiencies are rare but serious diseases with diverse genetic causes. Accumulating evidence suggests that defects in DNA double-strand break (DSB) repair can underlie many of these syndromes. In this context, the nonhomologous end joining pathway of DSB repair is absolutely required for lymphoid development, but possible roles for the homologous recombination (HR) pathway have remained more controversial. While recent evidence suggests that HR may indeed be important to suppress lymphoid transformation, the specific relationship of HR to normal lymphocyte development remains unclear. We have investigated roles of the X-ray cross-complementing 2 (Xrcc2) HR gene in lymphocyte development. We show that HR is critical for normal B-cell development, with Xrcc2 nullizygosity leading to p53-dependent early S-phase arrest. In the absence of p53 (encoded by Trp53), Xrcc2-null B cells can fully develop but show high rates of chromosome and chromatid fragmentation. We present a molecular model wherein Xrcc2 is important to preserve or restore replication forks during rapid clonal expansion of developing lymphocytes. Our findings demonstrate a key role for HR in lymphoid development and suggest that Xrcc2 defects could underlie some human primary immunodeficiencies.

Modeling the Spatial Distribution of Crimean-Congo Hemorrhagic Fever Outbreaks in Turkey

The more than 1100 human cases of Crimean-Congo Hemorrhagic Fever (CCHF) reported in a continuing series of outbreaks that started in 2003 in Turkey constitute the largest epidemic of the disease since CCHF was first recognized in 1944. The spatial distribution of CCHF case reporting rates in Turkey was studied to look for clusters of disease. We applied the spatial scan analysis to test the hypothesis of whether there were areas with a higher than expected number of CCHF cases. The analysis was conducted through windows of 10, 20, 40 and 80 km in diameter[SR1] to determine whether clustering of cases was dependent on the size of the scanning window. At the largest window size, consistent patterns of significantly higher than expected numbers of CCHF cases were found in a total of 40 administrative districts. A predictive model to map the habitat suitability for the vector tick was developed from satellite-based climate data and high-resolution features of the vegetation from Landsat images covering the whole country. It was found that areas of higher risk (higher CCHF reporting) were correlated (p<0.05) with zones of high climate suitability for the tick together with a high rate of fragmentation of agricultural land interspersed between forest and shrub-type vegetation.

60 IMPROVEMENT IN EFFICIENCY AND QUALITY OF CLONED AND PARTHENOGENETIC PORCINE EMBRYOS BY AMINO ACIDS

In vitro production of cloned porcine embryos by somatic cell nuclear transfer (SCNT) has become routine in several laboratories but the efficiency and quality of the resultant blastocysts remains sub-optimal. Cloned porcine blastocysts show low cell number, high fragmentation rate, and apoptosis which results in lower pregnancy rates upon embryo transfer. Earlier we reported that supplementation of culture media with amino acids benefit pre-implantation embryo development of in vivo- as well as in vitro-fertilized porcine embryos (Koo et al. 1997 Theriogenology 48, 791–802). This study evaluated how exogenous amino acids could affect pre-implantation development and quality of cloned or parthenogenetic porcine embryos. The effects of commercially available amino acids, referred to as Eagle&amp;apos;s non-essential amino acids (NEAA), added or not added (control) to NCSU23 medium containing fatty acid-free BSA were studied. Oocytes recovered from abattoir-derived prepubertal porcine ovaries were matured in vitro and parthenogenetically activated (PA) or nuclear-transferred with fetal fibroblasts (SCNT), as described earlier (Uhm et al. 2000 Mol. Reprod. Dev. 57, 331–337). At 168 h post-activation, blastocysts were harvested for assessment of embryo quality by TUNEL labeling, Hoechst 33342 staining, and gene expression analysis. Results showed that, in the PA group, the cleavage rate was not affected by the supplementation of NEAA. However, the blastocyst rate was significantly improved when NEAA was present in the medium compared to that of the control group (38.9 &amp;plusmn; 0.3 vs. 27.5 &amp;plusmn; 0.3&amp;percnt;, respectively) throughout the culture period. The supplementation during the pre-compaction period alone gave better results than during the post-compaction period alone (59.5 &amp;plusmn; 0.9 vs. 33.4 &amp;plusmn; 0.3&amp;percnt;, respectively). In the SCNT group, however, both cleavage (73.6 &amp;plusmn; 0.2 vs. 64.2 &amp;plusmn; 0.4&amp;percnt;) and blastocyst rate (18.7 &amp;plusmn; 0.2 vs. 13.8 &amp;plusmn; 0.3&amp;percnt;) were improved by NEAA supplementation. Furthermore, these blastocysts had higher hatching ability (30.0 &amp;plusmn; 1.8 vs. 14.6 &amp;plusmn; 4.9&amp;percnt;) than those of control group (P &amp;lt; 0.05). Supplementation of NEAA also increased the mean nuclei number of PA-derived (76.1 &amp;plusmn; 4.9 vs. 66.5 &amp;plusmn; 3.3) as well as SCNT-derived (43.1 &amp;plusmn; 2.6 vs. 31.8 &amp;plusmn; 1.9) blastocysts and reduced the time during which blastocysts formed. TUNEL assay revealed that incidence of nuclear fragmentation and apotosis was reduced by NEAA. Real-time qRT-PCR for Bax and Bcl-XL transcripts revealed that the relative abundance of Bax was reduced while that of Bcl-XL was increased. These effects were more pronounced when NEAA was present during the pre-compaction period alone. Thus, our data suggest that NEAA improves the yield and quality of cloned porcine embryos by enhancing blastocyst expansion and positively modulating the total cell number and apoptosis. These data may have implications for understanding the nutritional needs of cloned porcine embryos produced in vitro and for optimizing the composition of culture media to support their development. This work was supported by the Research Project on the Production of Bio-Organs (No. 200503030201), Ministry of Agriculture and Forestry, Republic of Korea.

Lipid imaging by gold cluster time-of-flight secondary ion mass spectrometry: application to Duchenne muscular dystrophy

Imaging with time-of-flight secondary ion mass spectrometry (TOF-SIMS) has expanded very rapidly with the development of gold cluster ion sources (Au(3+)). It is now possible to acquire ion density maps (ion images) on a tissue section without any treatment and with a lateral resolution of few micrometers. In this article, we have taken advantage of this technique to study the degeneration/regeneration process in muscles of a Duchenne muscular dystrophy model mouse. Specific distribution of different lipid classes (fatty acids, triglycerides, phospholipids, tocopherol, coenzyme Q9, and cholesterol) allows us to distinguish three different regions on a mouse leg section: one is destroyed, another is degenerating (oxidative stress and deregulation of the phosphoinositol cycle), and the last one is stable. TOF-SIMS imaging shows the ability to localize directly on a tissue section a great number of lipid compounds that reflect the state of the cellular metabolism.