DNA purification using dynamic solid-phase extraction on a rotationally-driven polyethylene-terephthalate microdevice.

We report the development of a disposable polyester toner centrifugal device for semi-automated, dynamic solid phase DNA extraction (dSPE) from whole blood samples. The integration of a novel adhesive and hydrophobic valving with a simple and low cost microfabrication method allowed for sequential addition of reagents without the need for external equipment for fluid flow control. The spin-dSPE method yielded an average extraction efficiency of ∼45% from 0.6 µL of whole blood. The device performed single sample extractions or accommodate up to four samples for simultaneous DNA extraction, with PCR-readiness DNA confirmed by effective amplification of a ß-globin gene. The purity of the DNA was challenged by a multiplex amplification with 16 targeted amplification sites. Successful multiplexed amplification could routinely be obtained using the purified DNA collected post an on-chip extraction, with the results comparable to those obtained with commercial DNA extraction methods. This proof-of-principle work represents a significant step towards a fully-automated low cost DNA extraction device.

Development of an animal model for prostate cancer cell metastasis to adult human bone.

BACKGROUND: Prostate cancer metastases to bone are associated with significant morbidity and mortality. Presently, there is little known about the biological interaction between prostate cancer cells and bone. Development of an animal model using adult human bone will enhance our ability to study the biology of prostate cancer metastasis to bone. METHODS: Bone was harvested from patients undergoing total joint arthroplasty and implanted in the hindlimbs of pre-treated SCID mice. Two months after bone implantation 4 x 104 prostate cancer cells (PC-3 or LAPC-4) were injected near the bone implantation site. The animals were sacrificed approximately 8 to 12 weeks after the injections of the cells. Complete histological analysis including immunostaining was performed. RESULTS: Both the PC-3 and LAPC-4 prostate cancer cells homed to the human bone implant, specifically the reconstituted bone marrow cavity. Analysis of the bone-tumor interaction after injection of PC-3 cells revealed strong labeling for PTHrP, TNF alpha and IL-6, consistent with osteoclast recruitment and osteoclast activity. These cells also were positively stained for CK18. After cellular injection of LAPC-4 cells, there was strong labeling for TNF alpha, IL-6, and IL-1 (osteoclast recruitment and osteolytic activity). PTHrP staining was also noted. The bone cells were strongly stained for osteocalcin, and the tumor cells for PSA. CONCLUSIONS: These data suggest that the tumor cells may induce an osteolytic response to enhance their ability to metastasize to bone. This animal model allows us to study the biologic interaction between prostate cancer cells and human bone and may enhance our understanding of the events associated with prostate cancer metastasis to bone.

Nuclear import of histone H2A and H2B is mediated by a network of karyopherins.

The first step in the assembly of new chromatin is the cell cycle-regulated synthesis and nuclear import of core histones. The core histones include H2A and H2B, which are assembled into nucleosomes as heterodimers. We show here that the import of histone H2A and H2B is mediated by several members of the karyopherin (Kap; importin) family. An abundant complex of H2A, H2B, and Kap114p was detected in cytosol. In addition, two other Kaps, Kap121p and Kap123p, and the histone chaperone Nap1p were isolated with H2A and H2B. Nap1p is not necessary for the formation of the Kap114p-H2A/H2B complex or for import of H2A and H2B. We demonstrate that both histones contain a nuclear localization sequence (NLS) in the amino-terminal tail. Fusions of the NLSs to green fluorescent protein were specifically mislocalized to the cytoplasm in kap mutant strains. In addition, we detected a specific mislocalization in a kap95 temperature-sensitive strain, suggesting that this Kap is also involved in the import of H2A and H2B in vivo. Importantly, we show that Kap114p, Kap121p, and Kap95 interact directly with both histone NLSs and that RanGTP inhibits this association. These data suggest that the import of H2A and H2B is mediated by a network of Kaps, in which Kap114p may play the major role.

Publication of abstracts submitted to the annual meeting of the Pediatric Orthopaedic Society of North America.

A computerized MEDLINE search was performed to determine the publication pattern of the abstracts submitted for podium presentation at the 1991-1994 annual meetings of the Pediatric Orthopaedic Society of North America (POSNA). The publication percentage for all papers submitted to the POSNA meetings from 1991 through 1994 was 45%. Fifty-three percent of papers accepted for podium presentation were ultimately published in comparison with 38% of those not accepted for presentation (p < 0.001). The mean time to publication was 29 months and did not differ significantly for the two groups. The majority of papers (65%) were published in either Journal of Pediatric Orthopaedics (48%) or The Journal of Bone and Joint Surgery (American) (17%). The frequency of ultimate publication of abstracts submitted to the annual POSNA meetings compares favorably with the rates for other medical subspecialties.

Publication rate of abstracts presented at the annual meeting of the Orthopaedic Research Society.

Although the timely conveyance of information at national meetings like those of the Orthopaedic Research Society is critical to the dissemination of new scientific research, the ultimate goal of most researchers is to publish work in peer-reviewed journals referenced in Medline. All of the abstracts that were presented at the podium at the 1991, 1992, and 1993 annual meetings of the Orthopaedic Research Society and printed in the appropriate yearly transactions were included in this study (n=888,296 per year). A detailed computerized Medline search of each author on the abstract and the appropriate keywords from the title was performed until a publication was found; otherwise, the abstract was excluded. The database was searched through June 30, 1997. A total of 463 (52%) of the abstracts were published by July 1, 1997. The percentages for each individual year were similar: 148 (50%) were published in 1991, 162 (55%) in 1992, and 153 (52%) in 1993. Publication of the majority of these papers (93.1%, 431 of 463) occurred within 4 years of the respective meeting. The Journal of Orthopaedic Research published the majority of these papers (17.5%), followed by The Journal of Bone and Joint Surgery (American), the Journal of Biomechanics, Clinical Orthopaedics and Related Research, and Spine (each 5.2%). No significant differences in the rate of publication were observed between papers of 10 broad subject categories (p=0.103). These results are similar to those from other basic science meetings and to the recently reported results for the annual meeting of the American Academy of Orthopaedic Surgeons.

Factors associated with active, refractory epistaxis.

This study addresses the underlying causes responsible for the severity and persistence of active, refractory epistaxis. Seventy-five patients referred because of treatment failure by primary care physicians showed hypertension and aspirin and alcohol abuse to be major factors in the refractory nature of their epistaxis. The majority of bleeding was located near the posterior floor of the nasal cavity and just posterior to Kiesselbach's plexus and was only associated with septal deviation, spurring, or mucosal abnormalities in 16 of the 75 patients. Seventeen of 67 outpatients required hospitalization. Standard laboratory tests were often inadequate determinants of etiology. Intractable epistaxis should be a signal for a thorough investigation of factors that influence clotting.