Silica-based colloid centrifugation enhances sperm quality in cockerel semen.

1. PercollTM is one of the most widely used colloid for animal sperm preparation. The aim of this study was to evaluate whether PercollTM colloid centrifugation could be practical to improve cockerel sperm quality, and to compare the effects of PercollTM single layer centrifugation (SLC) and density gradient centrifugation (DGC) in order to obtain the most optimal protocol for cockerel semen.2. In the experiment with PercollTM SLC for fresh semen, an increase of motile sperm was seen after PercollTM 80% SLC and 90% SLC was conducted, at levels of 28.8% and 30.2% respectively (P < 0.01). The increase of progressively motile sperm after PercollTM 80% SLC and 90% SLC was 177.2% and 202.4% respectively (P < 0.01). Meanwhile, for semen stored at 4°C for 24 h, the increase of motile sperm after PercollTM 70% SLC and 80% SLC was 41.2% and 44.0% (P < 0.01), and the increase of progressive sperm after PercollTM 70% SLC and 80% SLC was 71.3% and 83.1% respectively (P < 0.01). Both the percentage of motile sperm and progressive sperm of the fresh and stored cockerel semen after appropriate PercollTM SLC was significantly enhanced.3. Sperm membrane integrity did not show any decrease after PercollTM centrifugation compared with non-centrifuged semen, which suggested that the PercollTM centrifugation treatment in this study did not cause damage to cockerel sperm membranes.4. In the experiment regarding the comparison of PercollTM SLC and DGC with fresh semen, the increase of motile sperm after PercollTM 80% SLC, 90% SLC and 40%/80% DGC was 29.5%, 36.4%, and 25.0% respectively; and the increase of progressive sperm was 44.7%, 58.5%, and 54.7%, respectively. For semen stored at 4°C for 24 h, the increase of motile sperm after PercollTM 70% SLC, 80% SLC and 35%/70% DGC were 41.2%, 44.0%, and 26.4%; and the increase of progressive sperm was 71.3%, 83.1%, and 43.7%, respectively. There were no significant differences between the increase of sperm motility after PercollTM 80%, 90% SLC or PercollTM 40%/80% DGC in fresh cockerel semen. There was no significant difference between PercollTM 70%, 80% SLC and PercollTM 35%/70% in stored cockerel semen. There was a tendency for sperm recovery rates with PercollTM SLC to be higher than PercollTM DGC, although this did not reach statistical significance in this study.5. It was concluded that PercollTM SLC was more suitable for cockerel sperm separation than PercollTM DGC. The results suggested that PercollTM 80% SLC was the most optimal procedure to separate fresh cockerel sperm and PercollTM 70% SLC was the most optimal procedure to separate stored cockerel sperm. PercollTM SLC is more simple, user-friendly and economical and less time-consuming than DGC for cockerel semen processing.

[Study on the relationships between precollege sexual abuse experiences at different stages and adult attachment in college students].

Objective: To explore the association between precollege sexual abuse experiences at different periods and adult attachment in college students. Methods: In October 2018,a total of 4 034 college students were selected from 4 colleges in Hefei City by using the stratified cluster sampling method. A self-designed questionnaire was used to investigate the demographic characteristics, sexual abuse experience, and attachment style of participants. Multivariate linear regression analysis was performed to analyze the association between different periods, types of precollege sexual abuse experiences and attachment style in college students. Results: The rate of total precollege sexual abuse, noncontact sexual abuse, and contact sexual abuse was 14.1% (569), 12.7% (512) and 5.4% (219), respectively. Multivariate linear regression analysis showed that secure attachment [ß(95%CI)=-0.205 (-0.292, -0.117)] was negatively correlated with total precollege sexual abuse experiences, whereas anxiety attachment [ß(95%CI)=0.211 (0.110, 0.310)], avoidant attachment [ß(95%CI)=0.117 (0.020, 0.216)] were positively correlated with total precollege sexual abuse experiences. The number of periods of precollege noncontact sexual abuse was negatively correlated with secure attachment [ß(95%CI)=-0.106(-0.171, -0.041)]and positively correlated with anxiety attachment [ß(95%CI)=0.158 (0.084, 0.231)]and avoidant attachment [ß(95%CI)=0.080 (0.008, 0.152)]. The noncontact [ß(95%CI)=0.427 (0.018, 0.775)] and contact sexual abuse [ß(95%CI)=0.468 (0.251, 0.687)] in high school were positively correlated with anxiety attachment (P<0.05). Conclusion: Different periods, types of precollege sexual abuse experiences were all associated with adult attachment in college students.

Evaluation of cockerel spermatozoa viability and motility by a novel enzyme based cell viability assay.

1. The results of spermatozoa assessment by the WST-8 (2-[2-methoxy-4-nitrophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H-tetrazolium, monosodium salt) assay, flow cytometry (FC) or computer-assisted sperm analysis (CASA) were compared. 2. Different live/killed ratios of cockerel semen were serially diluted to 120, 60, and 30 × 106 cells/ml, and each sample was analysed by (1) WST-8 assay at 0, 10, 20, 30, 40, 50, 60 min, (2) viability with FC, and (3) motility with CASA. 3. The WST-8 reduction rate was closely correlated with spermatozoa viability and motility. The optimal semen concentration for the WST-8 assay was 120 × 106 cells/ml, and the standard curves for spermatozoa viability and motility predictions, respectively, were yviability60 = 162.8x + 104.96 (R2 = 0.9594) after 60 min of incubation and ymotility40 = 225.09x + 96.299 (R2 = 0.8475) after 40 min of incubation. 4. It was concluded that the WST-8 assay is useful for the practical evaluation of cockerel spermatozoa viability and motility. Compared to FC and CASA, the WST-8 assay does not require expensive and complex instrumentation in the lab. Furthermore, one well of the WST-8 reaction can be used to predict spermatozoa viability and motility at the same time, which all lead it to be efficient and economical for semen quality assessment.

[A clinicopathological classification analysis of ocular mass lesions in 7 910 cases].

Objective: To investigate the anatomical region, histopathological classification and histogensis distribution of ocular mass lesions in South China. Methods: Retrospective cases study. The clinical and pathological data of 7 910 samples with ocular (adnexal) tumors or proliferative lesions which were examined from January 2000 to May 2018 were retrospectively retrieved. The constituent ratios of ocular mass lesions in different anatomical regions and histogenesis have been analyzed. Results: There were 3 445 males and 4 465 females aged from 3 months to 106 years. Classification by anatomical region. Eyelid 4 976 cases (62.9%): benign-pigmented nevus (31.7%, 1 342/4 235), squamous cell papilloma (12.3%, 519/4 235), seborrheic keratosis (9.4%, 396/4 235); malignant-basal cell carcinoma (48.5%, 359/741), sebaceous gland carcinoma (34.4%, 255/741), squamous cell carcinoma (12.3%, 91/741). Ocular surface 1 449 cases (18.3%): benign-pigmented nevus (26.6%, 359/1 348), squamous cell papilloma (12.8%, 173/1 348); malignant-lymphoma (34.7%, 35/101), squamous cell carcinoma (30.7%, 31/101).Orbit 1 485 cases (18.8%): benign-hemangioma (28.5%, 332/1 167), lacrimal gland (duct) cyst(13.2%, 154/1 167); malignant-lymphoma (44.7%, 142/318), adenoid cystic carcinoma (10.1%, 32/318). Classification by histogenesis: epithelial 2 145 cases (27.1%), cutaneous appendages 378 cases (4.8%), cystoid 1 068 cases (13.5%), mesenchymal 748 cases (9.5%), lymph-hematopoietic 225 cases (2.8%), neurogenic 31 cases (0.4%), melanocytic 1 765 cases (22.3%), others 1 550 cases (19.6%). Conclusions: Over the past 18 years, the ocular tumors identified at the Second Affiliated Hospital, Zhejiang University School of Medicine most frequently occur in eyelid and originate from epithelium. The most common types are as followings. Benign lesions: pigmented nevus, squamous cell papilloma are the most common types for eyelid and ocular surface, whereas hemangioma, lacrimal gland (duct) cyst and epidermoid cyst are the most common types for orbit. Malignant cancers: basal cell carcinoma is the most prevalent disease in eyelid, whereas lymphoma occurs more frequently in ocular surface and orbit. (Chin J Ophthalmol, 2019, 55: 847-853).

Label swapper device for spectral amplitude coded optical packet networks monolithically integrated on InP.

In this paper the design, fabrication and experimental characterization of an spectral amplitude coded (SAC) optical label swapper monolithically integrated on Indium Phosphide (InP) is presented. The device has a footprint of 4.8x1.5 mm2 and is able to perform label swapping operations required in SAC at a speed of 155 Mbps. The device was manufactured in InP using a multiple purpose generic integration scheme. Compared to previous SAC label swapper demonstrations, using discrete component assembly, this label swapper chip operates two order of magnitudes faster.

Exploring the relationship between preterm placental calcification and adverse maternal and fetal outcome.

OBJECTIVES: To explore the relationship between preterm placental calcification and adverse pregnancy outcome, including maternal and fetal outcomes. METHODS: In this prospective cohort study, monthly ultrasonography was performed starting at 28 weeks' gestation to establish the diagnosis of Grade III placental calcification. Women were classified into three groups: Group 1, the early preterm group, with placental calcification found prior to 32 weeks (n = 63); Group 2, the late preterm group, with placental calcification found between 32 and 36 weeks (n = 192); and Group 3, the control group, without placental calcification noted between 28 and 36 weeks (n = 521). Women who smoked cigarettes or drank alcohol during pregnancy, or who had hypertension, diabetes, significant antenatal anemia or placenta previa were all excluded. Logistic regression analysis was used to estimate the risks of adverse pregnancy outcome in Groups 1 and 2 by calculating odds ratios (OR) with 95% CIs, adjusted by maternal age, body mass index, economic status, marital status, type of delivery and parity. RESULTS: Risks for adverse maternal outcome including postpartum hemorrhage (OR, 3.43; 95% CI, 1.251-9.388), placental abruption (OR, 6.52; 95% CI, 1.356-31.382) and maternal transfer to the intensive care unit (OR, 9.76; 95% CI, 1.826-52.195) and for adverse fetal outcomes including preterm birth (OR, 4.20; 95% CI, 1.775-9.940), low birth weight (OR, 4.58; 95% CI, 2.201-9.522), low Apgar score (OR, 6.53; 95% CI, 2.116-20.142) and neonatal death (OR, 9.04; 95% CI, 1.722-47.411) were much higher in Group 1 than in Group 3. In contrast, there were no significant differences in adverse pregnancy outcome between Groups 2 and 3. CONCLUSIONS: Early preterm placental calcification is associated with a higher incidence of adverse pregnancy outcome, and may serve as an indicator of adverse maternal and fetal outcomes when noted on ultrasonography. Conversely, women with late preterm placental calcification are not at greater risk for adverse pregnancy outcome.

An interval-based regret-analysis method for identifying long-term municipal solid waste management policy under uncertainty.

In this study, an interval-based regret-analysis (IBRA) model is developed for supporting long-term planning of municipal solid waste (MSW) management activities in the City of Changchun, the capital of Jilin Province, China. The developed IBRA model incorporates approaches of interval-parameter programming (IPP) and minimax-regret (MMR) analysis within an integer programming framework, such that uncertainties expressed as both interval values and random variables can be reflected. The IBRA can account for economic consequences under all possible scenarios associated with different system costs and risk levels without making assumptions on probabilistic distributions for random variables. A regret matrix with interval elements is generated based on a matrix of interval system costs, such that desired decision alternatives can be identified according to the interval minimax regret (IMMR) criterion. The results indicate that reasonable solutions have been generated. They can help decision makers identify the desired alternatives regarding long-term MSW management with a compromise between minimized system cost and minimized system-failure risk.

Large-scale mitochondrial DNA deletions in weak goslings.

1. Evidence has accumulated in mammals to support the idea that mitochondrial DNA (mtDNA) deletions and mutations might contribute to ageing and reproductive failure. White Roman geese were monitored to evaluate the effect of large-scale deletions of mtDNA in an avian species. 2. A total of 340 samples from 114 dead embryos, 111 weak goslings, and 115 normal goslings were used in this experiment. The regions of these two large-scale mtDNA deletions, ΔmtDNA6829 and ΔmtDNA6992, were between the COI and ND5 genes. A 3·6% (4 out of 111) positive sample was detected in the weak goslings. In contrast, no large-scale mitochondrial DNA deletions were detected in either the dead embryos (0 out of 114) or the normal goslings (0 out of 115). 3. Large-scale mtDNA deletions may be a factor causing weak goslings.

The origin of the white Roman goose.

In order to avoid interference from nuclear copies of mitochondrial DNA (numts), mtDNA of the white Roman goose (domestic goose) was extracted from liver mitochondria. The mtDNA control region was amplified using a long PCR strategy and then sequenced. Neighbor-joining, maximum parsimony, and maximum-likelihood approaches were implemented using the 1,177 bp mtDNA control region sequences to compute the phylogenetic relationships of the domestic goose with other geese. The resulting identity values for the white Roman geese were 99.1% (1,166/1,177) with western graylag geese and 98.8% (1,163/1,177) with eastern graylag geese. In molecular phylogenetic trees, the white Roman goose was grouped in the graylag lineage, indicating that the white Roman goose came from the graylag goose (Anser anser). Thus, the scientific name of the white Roman goose should be Anser anser 'White Roman.'

Output characterization of a self-pulsating and aperiodic optical fiber source based on cascaded regeneration.

We investigate the properties of a self-pulsating fiber cavity based on cascaded regeneration. The mechanisms that govern the number of oscillating pulses in the cavity, the pulse peak power, the pulse width, the wavelength tunability as well as the generation of sub-picosecond pulses are identified, analyzed and quantified. We find that the described self-pulsating cavity enables the oscillation of quasi transform-limited pulses with a pulsewidth of 4.8 ps at 1540.0 nm when using 0.4 nm non-Gaussian bandpass filters. Sub-picosecond pulses with an autocorrelation width of 471 fs are generated from the same self-pulsating source with modified bandpass filters and the addition of a chromatic dispersion compensator. The number of eigenpulses that oscillate simultaneously in the cavity can be adjusted from 0 up to 29,500 with proper cavity adjustment. This source has dual-wavelength output and can be tuned throughout the gain band of the amplifiers.

Establishment of the long-term in vitro culture system for chicken primordial germ cells.

The objective of this study was to establish the long-term in vitro culture system for chicken gonadal primordial germ cells (gPGCs). Primitive gonads collected from 5.5-day-old chicken embryos were dissociated and explanted onto plates pre-coated with 0.1% gelatin. Each of the four different conditioned media from proliferating and mitotically inactivated chicken embryonic fibroblast (CEF) cells and murine embryonic fibroblasts (STO cells, CRL-1053, ATCC, USA), respectively, was supplemented with growth factors and used to support the growth of gPGCs. The result showed that all the conditioned media could promote the growth and colony formation of gPGCs in vitro, in particular the medium conditioned by inactivated CEF cells. The gPGC-derived colonies maintained in inactivated CEF cells-conditioned medium up to 281 days were positively stained by periodic acid Schiff reaction and antibodies specific to anti-SSEA-1, SSEA-3, SSEA-4, integrin alpha6 and integrin beta1. Their capacities of migration via vascular system and taking up residence in the primary gonadal ridge were further demonstrated by transferring to the dorsal aorta of stage 17 recipient embryos. These results suggested that our culture system is able to maintain chicken gPGCs for long-term in vitro culture without losing their capacity to express pluripotent markers and to integrate into the gonads.

[Association between suffering experiences from precollege peer bullying at different stages and the quality of health-related life among college students].

Objective: To explore the effects of precollege peer bullying at different stages, on quality of life (QOL) among college students. Methods: A stratified cluster sampling method was used to select participants in October, 2018. Cluster sampling method was adopted to recruit a sample of 4 034 college students from four universities in Hefei city, Anhui province. Relations between peer bullying at different stages before entering college, and the quality of life, were investigated. t test and analysis of variance were used to compare the differences of QOL in different groups. Multiple linear regression models were used to assess the associations between various types of peer bullying at different stages and the QOL at precollege days. Results: Among all the 4 034 college students under study, mean scores of the 4 dimensions of QOL appeared as physical (12.61±2.02), psychological (14.09±2.62), social relationship (13.72±2.71) and environment (13.77±2.46), respectively. After adjusting the confounding factors, data from multiple linear regression showed that, factors as verbal bullying victimization during primary school (ß=-0.21)/secondary school (ß=-0.27)/or at both periods (ß=-0.56), relational (ß=-0.21) and physical (ß=-0.38) bullying victimization in secondary school, and physical bullying victimization in both periods (ß=-0.67) were negatively correlated with the physical dimension of QOL. Verbal bullying victimization in both periods (ß=-0.41) and relational bullying victimization in secondary school (ß=-0.42) were negatively correlated with psychological dimension of QOL. Factors as relational (ß=-0.32) and physical (ß=-0.51) bullying victimization in secondary school, physical/cyber bullying victimization in both periods (ß=-0.57) were negatively correlated with the social dimension of QOL. Verbal bullying victimization during primary school (ß=-0.20) and both periods (ß=-0.46), relational bullying perpetration during primary school (ß=-0.35) or at both periods (ß=-0.90) were negatively correlated with the environmental dimension of QOL (All P<0.05). Index related to bullying victimization (ß=-0.33, -0.36, -0.30, -0.33) and bullying perpetration ß=-0.28, -0.31, -0.23, -0.28) were both negatively correlated to all the 4 dimensions (physical, psychological, social relationship and environment) of QOL (P<0.001). Conclusions: Various forms of peer bullying experiences occurring before college, were associated with the decreased scores of QOL in different domains, among the university students. Programs on prevention and control of peer bullying in different stages before college days seemed important thus should be strengthened.

Successful induction of antisera against rabbit embryos for isolation of the ICM and putative embryonic stem cells.

In Expt 1, goat antisera against rabbit blastocysts were induced using spleen cell injection and skin-graft for immunosurgical isolation of ICM cells. Goats received rabbit spleen cell suspension (4 x 10(8) cells/ml) intravenously once a week for three consecutive weeks, plus an additional dose (boost injection) 10 days after the third injection, or a piece of rabbit skin (3 x 3 cm) transplantation. Blood samples were collected starting from the day after the last cell injection for 21 days. Serum was separated, heat inactivated and stored in frozen condition before titre analysis. Results showed that the antisera/antibodies derived by spleen cell injection reached their peak titre 7 days after the last cell injection, compared with 5 days by the skin-grafted group. In Expt 2, morphologically normal blastocysts were collected for isolating ICMs immunosurgically or for direct culture of zona-free whole blastocysts. In both methods, ICM cells started attaching to the feeder layer and outgrowing from the centre portion of the cells on day 3 after the onset of culture. ICM outgrowths increased in size during days 4-5, and most cells differentiated morphologically after day 6. One colony derived from isolated ICM developed into morphologically ES-like cells expressing alkaline phosphatase activity. Our results indicated that both skin-grafting and spleen cell injection were effective inducing antisera against rabbit embryonic cells. More studies are required to optimize the culture system for rabbit ES cells.

Texture evolution and mechanical behaviour of irradiated face-centred cubic metals.

A physically based theoretical model is proposed to investigate the mechanical behaviour and crystallographic texture evolution of irradiated face-centred cubic metals. This model is capable of capturing the main features of irradiated polycrystalline materials including irradiation hardening, post-yield softening and plasticity localization. Numerical results show a good agreement with experimental data for both unirradiated and irradiated stress-strain relationships. The study of crystallographic texture reveals that the initial randomly distributed texture of unirradiated metals under tensile loading can evolve into a mixture of [111] and [100] textures. Regarding the irradiated case, crystallographic texture develops in a different way, and an extra part of [110] texture evolves into [100] and [111] textures. Thus, [100] and [111] textures become dominant more quickly compared with those of the unirradiated case for the reason that [100] and [111]-oriented crystals have higher strength, and their plastic deformation behaviours are more active than other oriented crystals. It can be concluded that irradiation-induced defects can affect both the mechanical behaviour and texture evolution of metals, both of which are closely related to irradiation hardening.

In vitro and in vivo biological responses of plasma-sprayed hydroxyapatite coatings with posthydrothermal treatment.

This study was undertaken to evaluate the effect of post-hydrothermal treatment on the biological responses of the plasma-sprayed hydroxyapatite (HA)-coated Ti-6Al-4V implant system both in vitro and in vivo. After hydrothermal treatment, the HA coating (HAC) shows the high mechanical strength and indices-of-crystallinity, denser microstructure, lower concentrations of amorphous and impurity phases, when compared with the as-sprayed HAC. The in vitro cell-culture studies, using UMR106 osteoblast-like cell, demonstrated no signifiacnt cell growth on both surface of as-sprayed and hydrothermal-treated HACs during 10-day culture. The in vivo studies, using the transcortical implant model in the femora of goats, evaluated the histological responses of two coatings. After 6 week of implantation, using backscattered electron images, no substantial histological variations in the extents of new bone apposition and new bone healing between the two HACs were observed. However, the as-sprayed HAC, owing to the dissolution induced the granular particles dissociated from the HAC, showed the statically lower extent of new bone apposition than hydrothermal-treated HAC at 12 weeks. The results suggest that hydrothermal treatment could be used to improve the mechanical strength, crystallinity, and phase composition of HAC, which are important factors of long-term fixation and stability of implant. Besides, the treated HAC could also achieve the initial fixation of implant in clinical use.

The effect of electrical field strength on activation and development of cloned caprine embryos.

The activation procedure used in nuclear transfer (NT) is one of the critical factors affecting the efficiency of animal cloning. The purpose of this study was to compare the effect of two electrical field strengths (EFS) for activation on the developmental competence of caprine NT embryos reconstructed from ear skin fibroblasts of adult Alpine does. The NT embryos were obtained by transfer of the quiescent fibroblasts at the fourth passage into the enucleated metaphase II (M II) oocytes. Four to five hours after electrical fusion, the NT-embryos were activated by EFS either at 1.67 or at 2.33 kV/cm and immediately incubated in 6-DMAP (2 mM) for 4 h. The cleavage rate of the NT-embryos activated with 2.33 kV/cm was greater than that activated with 1.67 kV/cm after in vitro culture for 18 h (65.6% versus 19.6%, p < 0.001). No pregnancy was found in 14 recipient does after transferring 51 NT embryos at 1-2 cell stages activated with 1.67 kV/cm. In contrast, two of the seven recipients were pregnant and gave birth to three kids after transferring 61 NT embryos at 1-2 cell stages activated by 2.33 kV/cm. The birth weights of three cloned kids were within the normal range of Alpine goats. However, one kid died 1h after birth while the remaining two are still healthy. DNA analysis by polymerase chain reaction (single-strand conformation polymorphism, SSCP) confirmed that the three kids were genetically identical to the nuclear donor.

Effect of different sample pretreatment methods on the concentrations of excitatory amino acids in cerebrospinal fluid determined by high-performance liquid chromatography.

Using high-performance liquid chromatography (HPLC), the effect of different sample pretreatment methods on the concentrations of excitatory amino acids (EAAs, glutamate and aspartate) measured in cerebrospinal fluid (CSF) was investigated. The results showed that the measured values of glutamate and aspartate were constant when the samples were stored at -80 degrees C and then methanol was used for CSF deproteinization before assay; the values of glutamate (Glu) increased when 0.3 M perchloric acid was used for CSF deproteinization with the CSF subsequently being stored at -20 degrees C; the values of Glu changed when the samples were stored at -20 degrees C over 8 weeks with methanol subsequently being used for CSF deproteinization before assay. This reference data suggested that the CSF sample would be better stored at -80 degrees C. If the sample is stored at -20 degrees C over 8 weeks, the Glu values change with the storage time. If strong acidic reagents are used for precipitation of protein in the CSF sample and then stored at -20 degrees C, Glu values are abnormally increased. From this study, an accurate and sensitive reversed-phase high-performance liquid chromatographic method has been developed for anti-excitotoxicity therapy and thorough study of EAAs in a clinical setting.

Solid-state behavior of cromolyn sodium hydrates.

Cromolyn sodium (CS, disodium cromoglycate) is an antiasthmatic and antiallergenic drug. The solid-state behavior of CS is still not completely understood. CS forms nonstoichiometric hydrates and sorbs and liberates water in a continuous manner, although with hysteresis. The reported continuous changes in crystal lattice parameters of CS, which are associated with the changes in water stoichiometry, renders CS physically variable, which may complicate formulation and processing. In addition, controversies still remain as to whether CS exists as different stoichiometric hydrates, mainly because of its variable powder X-ray diffraction (PXRD) patterns (Cox, J. S. G. et al. J. Pharm. Sci. 1971, 60, 1458-65), which indicates a variable crystal structure. The objectives of this study are (a) to understand this unusual water uptake in the light of the molecular and crystal structures of CS, (b) to understand the relationship between the crystal structure and the PXRD patterns using Rietveld analysis, and (c) to investigate whether CS exists as different stoichiometric hydrates. The crystal structure of CS containing 6.44 molecules of water per molecule of CS was determined at 295 and 173 K. The packing arrangements in these structures (space group P1) are similar to those in a previous report, in which the water stoichiometry is 5 to 6, but the bond lengths, bond angles, and lattice parameters are different, reflecting the different water stoichiometries. In the crystal structure solved at 295 K, the position of only one of the two sodium ions could be determined. In the crystal structure solved at 173 K, the previously undetermined sodium ion is disordered over three sites, while four of eight water positions are partially occupied. The 2-hydroxy-propane chain that links the two cyclic moieties of CS was found to be flexible, perhaps allowing the CS crystal to accommodate variable amounts of water. The lack of a fixed coordination site for the second sodium ion may contribute to the disorder of the water molecules. The nonstoichiometric water content of CS is mainly attributed to the water molecules that are associated with the two unoccupied sodium sites. From the PXRD patterns of CS powder, equilibrated at various relative humidities, the various lattice parameters, including previously unreported alpha, beta, and gamma values, were calculated using Rietveld analysis. The peak shifts in these PXRD patterns are quantitatively explained by slight changes in the unit cell parameters. The recently described solid forms of CS were prepared and were found to correspond to the original crystalline CS, described by Cox et al. (1971), but with contamination by the known M mesophase in various proportions. The present results support a variable crystal structure and not the existence of different stoichiometric hydrates of CS.

Establishment of pluripotent cell lines from porcine preimplantation embryos.

Embryonic stem (ES) cells are pluripotent cells isolated from in vitro culture of preimplantation embryos. Experiments were undertaken to identify preimplantation embryonic stages and culture conditions under which pluripotent, porcine embryo-derived cell lines could be isolated. Cell lines were established from in vitro culture of intact, porcine early hatched blastocysts and isolated inner cell masses (ICM) from intermediate and late hatched blastocysts on feeder layers prepared from permanent mouse embryonic fibroblasts (STO). The cells of these porcine embryo-derived cell lines had a morphology similar to that of murine ES cells, but colony morphology was more epithelial-like. The cell lines retained a normal diploid karyotype, consistently expressed alkaline phosphatase activity, and survived cryopreservation. When subjected to in vitro differentiation, either spontaneous or induced, the embryo-derived cell lines differentiated extensively into a wide range of cell types representing the 3 embryonic germ layers. In vivo pluripotency of the cells was demonstrated by birth of a chimeric piglet, documented by pigmentation and DNA markers, and the ability to direct the development of nuclear-transfer embryos to the blastocyst stage. Such pluripotent embryo-derived cells provide a potential route for porcine genetic manipulation.