Methods for derivation of human embryonic stem cells

The expanded blastocysts, developed from 2PN-stage embryos, are generally divided into three categories: a good blastocyst containing a large and distinguishable inner cell mass (ICM), a blastocyst with a small and distinct ICM, and a blastocyst with a poorly defined ICM. In this study, we introduce methods for the derivation of human embryonic stem cells (hESCs) depending on the quality of the blastocysts. An immunosurgical method was used for the good expanded blastocysts. This method, however, raises the probability of ICM loss in cases of hESC derivation from blastocysts with smaller or indistinct ICMs. Furthermore, this method is also associated with a risk of the contamination of the hESCs with animal pathogens. To overcome these shortcomings, the partial- or whole-embryo culture method was used. For blastocysts with no visible ICM, the whole-embryo culture method was used to establish hESCs via the seeding of the entire blastocyst without its zona pellucida directly on a STO feeder layer. However, trophectodermal overgrowth tends to hinder the expansion of the ICM during the initial steps of hESC derivation. Therefore, the partial-embryo culture method was developed to establish hESCs from blastocysts with smaller ICMs. The surgical isolation of the region containing the ICM with an ultra-fine glass pipette alleviates trophectoderm overgrowth. This method is also applicable to blastocysts with large and distinct ICMs, and the efficiency of this method is comparable to that of the immunosurgical method.     

Emergency department visits for asthma: the role of frequent symptoms and delay in care.

BACKGROUND: Use of the emergency department (ED) for asthma care is a costly form of health care that is largely preventable. However, little is known about how to reduce the number of people using the ED for asthma care. OBJECTIVE: To identify modifiable factors related to ED visits for asthma among a diverse nonelderly adult population. METHODS: This study used cross-sectional data from the 2001 California Health Interview Survey. A total of 4,359 adult respondents ages 18 to 64 years who reported being diagnosed as having asthma and experiencing symptoms in the past year were included. Any ED visits due to asthma in the previous 12 months among all nonelderly respondents with asthma, with stratification by those with daily or weekly symptoms and with less frequent symptoms, were examined. RESULTS: Adults with daily or weekly asthma symptoms, with fair or poor health status, and who delayed care for asthma because of cost or insurance issues were more likely to visit the ED for asthma. Stratification of the study population into those with daily or weekly symptoms and those with less frequent symptoms revealed that delay in care due to cost or insurance issues and fair or poor health status remained significant for both groups. Latinos and women were more likely to visit the ED in the severe asthma group, whereas Asian, African American, and uninsured adults were more likely to visit the ED in the group with less severe asthma. CONCLUSIONS: Results suggest that to prevent ED visits for asthma, it is important to control asthma symptoms. However, it is equally if not more important to reduce delays in receiving asthma care.     

Biomimetic deposition of apatite coating on surface-modified NiTi alloy

TiO(2) coatings were prepared on NiTi alloy by heat treatment in air at 300, 400, 600 and 800 degrees C. The heat-treated NiTi alloy was subsequently immersed in a simulated body fluid for the biomimetic deposition of the apatite layer onto the surface of TiO(2) coating. The apatite coatings as well as the surface oxide layer on NiTi alloy were characterized using scanning electron microscopy equipped with energy dispersive spectroscopy, X-ray diffraction, X-ray photoelectron spectroscopy and Raman spectroscopy. Results showed the samples heat-treated at 600 degrees C produced a layer of anatase and rutile TiO(2) on the surface of NiTi. No TiO(2) was detected on the surface of NiTi after heat treatment at 300 and 400 degrees C by X-ray diffraction, while rutile was formed on the surface of the 800 degrees C heat-treated sample. It was found that the 600 degrees C heat-treated NiTi induced a layer consisted of microcrystalline carbonate containing hydroxyapatite on its surface most effectively, while 300 and 400 degrees C heat-treated NiTi did not form apatite. This was due to the presence of anatase and/or rutile in the 600 and 800 degrees C heat-treated NiTi which could provide atomic arrangements in their crystal structures suitable for the epitaxy of apatite crystals, and anatase had better apatite-forming ability than rutile. XPS and Raman results revealed that this apatite layer was a carbonated and non-stoichiometric apatite with Ca/P ratio of 1.53, which was similar to the human bone. The formation of apatite on 600 degrees C heat-treated NiTi following immersion in SBF for 3 days indicated that the surface modified NiTi possessed excellent bioactivity.     

Frequent allelic loss of 21q11.1 approximately q21.1 region in advanced stage oral squamous cell carcinoma

A fine mapping of loss of heterozygosity (LOH) was performed in oral squamous cell carcinoma (OSCC), using 12 markers on 21q11.1 approximately q21.1. We studied 43 resected primary invasive tumors and their paired normal tissues, concurrent dysplasia or carcinoma in situ in separate areas from 8 of the specimens, and 6 local recurrent carcinomas. LOH status was compared between lesions of different phases of progression within the same patient. A high frequency of LOH was observed for D21S1410, D21S120, and D21S1433 (60% each) in the primary lesions, constituting two interstitial deleted regions encompassing eight known genes. Cases showing LOH of D21S120 were significantly associated with advanced clinical stages (III and IV; P=0.02). Consistent allelic loss was observed in 64.2% of the informative cases between the precursor lesions and their corresponding invasive tumors, and in 59.5% of those between the primary lesions and their recurrent counterparts. Fewer than half of the different lesions within a given patient showed discordant allelic loss for tested markers. Our results suggest that 21q11.1 approximately q21.1 harbors tumor suppressor genes in OSCC. Genetic divergence may develop during tumor clone evolution.     

Geminin regulates neuronal differentiation by antagonizing Brg1 activity

Precise control of cell proliferation and differentiation is critical for organogenesis. Geminin (Gem) has been proposed to link cell cycle exit and differentiation as a prodifferentiation factor and plays a role in neural cell fate acquisition. Here, we identified the SWI/SNF chromatin-remodeling protein Brg1 as an interacting partner of Gem. Brg1 has been implicated in cell cycle withdrawal and cellular differentiation. Surprisingly, we discovered that Gem antagonizes Brg1 activity during neurogenesis to maintain the undifferentiated cell state. Down-regulation of Gem expression normally precedes neuronal differentiation, and gain- and loss-of-function experiments in Xenopus embryos and mouse P19 cells demonstrated that Gem was essential to prevent premature neurogenesis. Misexpression of Gem also suppressed ectopic neurogenesis driven by Ngn and NeuroD. Gem's activity to block differentiation depended upon its ability to bind Brg1 and could be mediated by Gem's inhibition of proneural basic helix-loop-helix (bHLH)-Brg1 interactions required for bHLH target gene activation. Our data demonstrate a novel mechanism of Gem activity, through regulation of SWI/SNF chromatin-remodeling proteins, and indicate that Gem is an essential regulator of neurogenesis that can control the timing of neural progenitor differentiation and maintain the undifferentiated cell state.     

Comparative evaluation of nonradiometric BACTEC and improved oxoid signal blood culture systems in a clinical laboratory.

The BACTEC NR660 blood culture system, which uses infrared spectroscopy to detect carbon dioxide generated by bacterial growth, was compared with the new medium formulation of the Oxoid Signal system. Two trials were conducted: a comparative study of 88 organisms in simulated blood cultures and a clinical trial of 3,321 paired patient blood culture samples. Both trials showed that overall the BACTEC system performed better in the recovery of organisms. The Oxoid system was unable to detect by signal the growth of the majority of yeasts, nonfermentative gram-negative bacilli, Neisseria meningitidis, Nocardia spp., and Corynebacterium jeikeium. There were no significant differences in the yield of Staphylococcus spp., members of the family Enterobacteriaceae, Streptococcus spp., or anaerobic organisms. BACTEC detected growth more quickly than did the Oxoid system; 61% of the isolates were detected by BACTEC at 24 h, while 49% of the isolates were detected by Oxoid. The Oxoid system had a high proportion (58.5%) of false-positives, compared with 7.7% for the BACTEC system. Despite the new medium formulation of the Oxoid system, its performance is still not equivalent to that of the BACTEC system.     

Transfer of immunity to cryptococcosis by T-enriched splenic lymphocytes from Cryptococcus neoformans-sensitized mice.

Splenic enriched T-cells and sera were obtained from inbred CBA/J mice injected 7 or 35 days earlier with either 10(3) viable Cryptococcus neoformans or sterile physiological saline. The transfer of enriched T-cells collected 7 days after immunization or of normal enriched T-cells did not transfer immunity to C. neoformans or delayed-type hypersensitivity responsiveness to cryptococcal culture filtrate (CneF) antigen to the recipients. However, enriched T-cells harvested 35 days after immunization, when transferred to recipient mice, were able to confer immunity as indicated by the reduction in numbers of C. neoformans cells in the tissues, and they also transferred delayed-type hypersensitivity responsiveness to CneF antigens. Sera from either sensitized or normal mice were unable to transfer immunity to recipient animals. These results suggested that there was a time requirement for development of the immune response in the donor mice and that T-cells were crucial in the host defense against a cryptococcal infection. Culturing of day-35 C. neoformans-sensitized T-cells in the presence of homologous antigen (CneF) but not in the presence of heterologous antigen (purified protein derivative or 2, 4-dinitro-1-fluorobenzene) induced the production of migration inhibition factor, thus indicating that lymphocytes from C. neoformans-injected mice were specifically sensitized to CneF antigen.