Breakthrough in purification of fossil pollen for dating of sediments by a new large-particle on-chip sorter.

Particle sorting is a fundamental method in various fields of medical and biological research. However, existing sorting applications are not capable for high-throughput sorting of large-size (>100 micrometers) particles. Here, we present a novel on-chip sorting method using traveling vortices generated by on-demand microjet flows, which locally exceed laminar flow condition, allowing for high-throughput sorting (5 kilohertz) with a record-wide sorting area of 520 micrometers. Using an activation system based on fluorescence detection, the method successfully sorted 160-micrometer microbeads and purified fossil pollen (maximum dimension around 170 micrometers) from lake sediments. Radiocarbon dates of sorting-derived fossil pollen concentrates proved accurate, demonstrating the method's ability to enhance building chronologies for paleoenvironmental records from sedimentary archives. The method is capable to cover urgent needs for high-throughput large-particle sorting in genomics, metabolomics, and regenerative medicine and opens up new opportunities for the use of pollen and other microfossils in geochronology, paleoecology, and paleoclimatology.

First Direct Mass Measurements of Nuclides around Z=100 with a Multireflection Time-of-Flight Mass Spectrograph.

The masses of ^{246}Es, ^{251}Fm, and the transfermium nuclei ^{249-252}Md and ^{254}No, produced by hot- and cold-fusion reactions, in the vicinity of the deformed N=152 neutron shell closure, have been directly measured using a multireflection time-of-flight mass spectrograph. The masses of ^{246}Es and ^{249,250,252}Md were measured for the first time. Using the masses of ^{249,250}Md as anchor points for α decay chains, the masses of heavier nuclei, up to ^{261}Bh and ^{266}Mt, were determined. These new masses were compared with theoretical global mass models and demonstrated to be in good agreement with macroscopic-microscopic models in this region. The empirical shell gap parameter δ_{2n} derived from three isotopic masses was updated with the new masses and corroborates the existence of the deformed N=152 neutron shell closure for Md and Lr.

In vitro measurement of tissue integrity during saccular aneurysm embolizations for simulator-based training.

In the domain of endovascular neurosurgery, the measurement of tissue integrity is needed for simulator-based training and for the development of new intravascular instruments and treatment techniques. In vitro evaluation of tissue manipulation can be achieved using photoelastic stress analysis and vasculature modeling with photoelastic materials. In this research we constructed two types of vasculature models of saccular aneurysms for differentiation of embolization techniques according to the respect for tissue integrity measurements based on the stress within the blood vessel model wall. In an aneurysm model with 5 mm dome diameter, embolization using MicroPlex 10 (Complex 1D, with 4 mm diameter loops), a maximum area of 3.97 mm² with stress above 1 kPa was measured. This area increased to 5.50 mm² when the dome was touched deliberately with the release mechanism of the coil, and to 4.87 mm² for an embolization using Micrusphere, (Spherical 18 Platinum Coil). In a similar way trans-cell stent-assisted coil embolization was also compared to human blood pressure simulation using a model of a wide-necked saccular aneurysm with 7 mm diameter. The area with stress above 1kPa was below 1 mm² for the pressure simulation and maximized at 3.79 mm² during the trans-cell insertion of the micro-catheter and at 8.92 mm² during the embolization. The presented results show that this measurement system is useful for identifying techniques compromising tissue integrity, comparing and studying coils and embolization techniques for a specific vasculature morphology and comparing their natural stress variations such as that produced by blood pressure.

Photoelastic stress analysis in blood vessel phantoms: three-dimensional visualization and saccular aneurysm with bleb.

BACKGROUND: The photoelastic effect is used for stress measurement during endovascular surgery simulation for quantitative evaluation of catheter trajectory in in vitro environments. By extending the capabilities of this sensing technology, its potential for intravascular tools evaluation will increase. METHODS: In this research the error introduced by stress direction on magnitude measurements was studied, then stress measurements were made in the phantom modelling of a saccular aneurysm with bleb. To visualize three-dimensionally the stress field changes produced by a guide wire in a phantom wall, a scanner and an algorithm relying on maximum likelihood-expectation maximization are proposed. Three-dimensional fields at different pressure level were compared with the stress field surrounding the guide wire. RESULTS: The maximum error in stress magnitude measurements due to stress direction was 2.52%. Stress local maximum was detected in the bleb phantom before rupture. Three-dimensional visualization was obtained in vasculature phantom with average errors of 10.73%, 4.55%, 3.18% for inner pressures of 80, 120, 160 mmHg, respectively. Stress measurement in the neighbourhood of the guide wire is equivalent to applying an inner pressure of 120 mmHg. CONCLUSIONS: For the presented polariscope, the weak influence of stress direction in magnitude measurements was confirmed. In vasculature phantoms, the three-dimensional visualization of stress eliminated birefringence visualization distortion and enabled more comprehensive comparison of stress produced by intravascular tools with stress produced by normal blood pressure.

Autonomous catheter insertion system using magnetic motion capture sensor for endovascular surgery.

BACKGROUND: In order to reduce fluoroscope usage in endovascular surgery, there is a need to develop autonomous catheter insertion systems. METHODS: We propose a system for tracking the position and speed of a catheter using a magnetic motion capture sensor to provide feedback to a catheter-driving mechanism, to perform autonomous catheter insertion in major vasculature. Catheter insertion speed control and path reconstruction experiments were performed with the system inside a silicone model of major vasculature to simulate surgery. RESULTS: The system controlled the catheter for speeds of 6.14 mm/s and reproduced a two-dimensional path inside the silicone blood vessel phantom with less than 7 mm of error. CONCLUSIONS: We found that error in speed control rises as a result of friction between the catheter and the model wall. Path reconstruction error depends on the model's cross-sectional diameter, the properties of the catheter insertion mechanism, the magnetic sensor and the system guidance technique.

Homing, proliferation and survival sites of human leukemia cells in vivo in immunodeficient mice.

The cellular components of the hematopoietic stem cell niche have been gradually identified. However, the niche for malignant hematopoiesis remains to be elucidated. Here, using human leukemia cells, which could be transplanted to immunodeficient mice, we studied the in vivo homing, proliferation and survival sites by immunohistopathology, compared with the corresponding sites for cord blood CD34(+) (CBCD34(+)) cells. The human leukemia cells initially localized on the surface of osteoblasts in the epiphysial region, and expanded to the inner vascular and diaphysial regions within 4 weeks. The percentage of CD34(+) leukemia cells in the bone marrow was transiently increased up to 50%. In vivo 5-bromo-2'-deoxyuridine labeling revealed that the epiphysis was the most active site for leukemia cell proliferation. CBCD34(+) cells showed the similar pattern of homing and proliferation to leukemia cells. After high-dose administration of cytosine-1-beta-D-arabinofuranoside, residual leukemia cells were localized in the perivascular endothelium as well as in contact with the trabecular endosteum. These findings suggest that xenotransplantation into immunodeficient mice provides a useful model to study the leukemia niche.

Bifurcation of osteoclasts and dendritic cells from common progenitors.

Osteoclasts and dendritic cells are derived from monocyte/macrophage precursor cells; however, how their lineage commitment is regulated is unknown. This study investigated the differentiation pathways of osteoclasts and dendritic cells from common precursor cells at the single-cell level. Osteoclastogenesis induced by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-kappaB ligand (RANKL) or tumor necrosis factor-alpha (TNF-alpha) is completely inhibited by addition of granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 at early stages of differentiation. GM-CSF-treated cells express both c-Fms and RANK and also low levels of CD11c and DEC205, which are detected on dendritic cells. Addition of GM-CSF also reduces expression of both c-Fos and Fra-1, which is an important event for inhibition of osteoclastogenesis. Overexpression of c-Fos by retroviral infection or induction in transgenic mice can rescue a failure in osteoclast differentiation even in the presence of GM-CSF. By contrast, differentiation into dendritic cells is inhibited by M-CSF, indicating that M-CSF and GM-CSF reciprocally regulate the differentiation of both lineages. Dendritic cell maturation is also inhibited when c-Fos is expressed at an early stage of differentiation. Taken together, these findings suggest that c-Fos is a key mediator of the lineage commitment between osteoclasts and dendritic cells. The lineage determination of osteoclast progenitors seen following GM-CSF treatment functions through the regulation of c-Fos expression.

ALCAM (CD166): its role in hematopoietic and endothelial development.

A critical role for the endothelium of yolk sac and dorsal aorta has been shown in embryonic hematopoiesis. A stromal cell line derived from yolk sac, YSCL-72, has been chosen to search for a novel molecule associated with embryonic hematopoiesis. Analysis between YSCL-72 and an adult aorta-derived endothelial cell line, EOMA, demonstrated that activated leukocyte cell adhesion molecule (ALCAM, or CD166) was specifically expressed in YSCL-72 but not in EOMA. Immunohistochemical study showed that ALCAM was expressed in the endothelium of yolk sac and dorsal aorta but not in adult aorta. ALCAM-transfected EOMA cells supported development of hematopoietic progenitor cells compared with vector-transfected EOMA cells, suggesting that ALCAM appeared to be crucial for hematopoiesis. In addition, ALCAM was found to be involved in capillary tube formation and hemangioblast differentiation. Taken together with these findings, ALCAM is highly associated not only with embryonic hematopoiesis but also vasculoangiogenesis.

Difference in quasispecies of the hypervariable region 1 of hepatitis C virus between alcoholic and non-alcoholic patients.

BACKGROUND: Habitual alcohol intake is known to aggravate the clinical outcome of hepatitis C virus (HCV)-related chronic liver diseases and to increase the risk of hepatocellular carcinoma. METHODS: To investigate the possible mechanism of these effects by alcohol, we examined 31 cases of HCV-related chronic liver diseases of which 17 cases were drinking just before admission and the remaining 14 cases were non-drinkers. The studied cases included 18 patients with chronic hepatitis, six with liver cirrhosis and seven with hepatocellular carcinoma. The quasispecies of the hypervariable region 1 of the HCV genome were analyzed by using polymerase chain reaction single strand conformation polymorphism (PCR-SSCP). Hepatitis C virus viral load was quantitated by using multicyclic PCR after reverse transcription of the 5' non-coding region of the genome. RESULTS: The mean PCR-SSCP band number that reflected the quasispecies complexity in hypervariable region 1 was more significantly increased in alcoholics than in non-alcoholics (5.5 +/- 1.4 vs 3.9 +/- 1.1, P< 0.01). The significant increase in alcoholics remained, even if the cases were restricted to males (P < 0.01), to HCV genotype 1b (P < 0.05) or to chronic hepatitis (P < 0.05). The HCV viral load was not statistically different between alcoholic and non-alcoholic HCV-related chronic liver diseases (5.02 x 10(6) +/- 5.16 x 10(6) copies/mL vs 9.00 x 10(7) +/- 2.75 x 10(8) copies/mL, P = 0.28). Mutation events seemed to occur randomly when amino acid sequences of hypervariable region 1 were compared between four drinkers and four non-drinkers. CONCLUSIONS: The enhanced quasispecies complexity in hypervariable region 1 of HCV in alcoholics may be the main cause of more progressive HCV-related chronic liver diseases, and may provide the disease the resistance against any therapeutic modalities including interferon.

High-speed separation system of randomly suspended single living cells by laser trap and dielectrophoresis.

We developed a new system for random separation of a single microorganism, such as a living cell and a microbe, in the microfluidic device under the microscope by integrating the laser-trapping force and dielectrophoretic (DEP) force. An arbitrarily selected single microbe could be isolated in a microchannel, despite the presence of a large number of microbes in solution. Once the target microbe is trapped at the focal point of the laser, we can easily realize exclusion of excess microbes around the target by controlling the electric field, while keeping the target trapped by the laser at the focal point. To realize an efficient separation system, we proposed a new separation cell and produced it by microfabrication. Flow speed in the microchannel is adjusted and balanced to realize high-speed and high-purity extraction of the target. Some preliminary experiments are conducted to show the effectiveness. The target is trapped by the laser, transported, and is taken out from the extraction port. Total separation time is less than 20 s. Our method is extremely useful in the pure cultivation of the cell and will be a promising method for biologists in screening useful microbes.

An intelligent catheter system robotic controlled catheter system.

SUMMARY: We have developed a novel catheter system, an intelligent catheter system, which is able to control a catheter by an externally-placed controller. This system has made from master-slave mechanism and has following three components; 1) a joy stick as a master (for operators) 2) a catheter controller as a slave (for a patient), 3) a micro force sensor as a sensing device. This catheter tele-guiding system has abilities to perform intravascular procedures from the distant places. It may help to reduce the radiation exposures to the operators and also to help train young doctors.

An adherent condition is required for formation of multinuclear osteoclasts in the presence of macrophage colony-stimulating factor and receptor activator of nuclear factor kappa B ligand.

Identification of receptor activator of nuclear factor-kappaB (RANK) and RANK-ligand (RANKL) has provided new insights into the osteoclast differentiation pathway. Osteoclast precursor cells were isolated using monoclonal antibodies against c-Fms and RANK, and the effect of adherence on the in vitro differentiation and proliferation of these cells was examined in 2 different types of stromal-cell-free culture systems: a semisolid culture medium (a nonadherent system) and a liquid culture medium (an adherent system). Osteoclast precursor cells were not able to differentiate into mature osteoclasts efficiently in the semisolid culture system. Trimerized RANKL enhanced osteoclast differentiation in semisolid cultures, but not to the extent seen when cells were allowed to adhere to plastic. Initial precursor cells were capable of differentiating into macrophages or osteoclasts. Once these cells were transferred to adherent conditions, striking differentiation was induced. Multinuclear cells were observed even after they had displayed phagocytic activity, which suggests that cell adhesion plays an important role in the differentiation of osteoclast precursor cells. Integrins, especially the arginine-glycine-aspartic acid (RGD)-recognizing integrins alpha(v) and beta(3), were needed for osteoclast-committed precursor cells to proliferate in order to form multinuclear osteoclasts, and the increase in cell density affected the formation of multinuclear cells. A model of osteoclast differentiation with 2 stages of precursor development is proposed: (1) a first stage, in which precursor cells are bipotential and capable of anchorage-independent growth, and (2) a second stage, in which the further proliferation and differentiation of osteoclast-committed precursor cells is anchorage-dependent. (Blood. 2000;96:4335-4343)

Effects of VEGF on Ca(2+)-transient in cultured lymphatic endothelial cells and mechanical activity of isolated lymph vessels.

We investigated the effects of vascular endothelial growth factor (VEGF(165)) on [Ca(2+)](i)-transient in cultured lymphatic endothelial cells (LEC) and mechanical activity of isolated dog thoracic ducts. VEGF (0.1-10 ng/ml) caused a dose-dependent increase of the [Ca(2+)](i) in LEC. Pretreatment with 10(-5) M genistein or 5x10(-6) M herbimycin A produced a significant reduction of the VEGF-induced [Ca(2+)](i)-transient. In the presence of 10(-6) M thapsigargin, VEGF caused no significant effect on the [Ca(2+)](i)-transient. Pretreatment with Ca(2+)-free solution containing 0.1 mM EGTA produced no significant effect on the peak increase of [Ca(2+)](i) induced by 0.1 or 10 ng/ml VEGF, but significantly depressed the sustained part of [Ca(2+)](i) observed at the higher concentration of VEGF. The VEGF (0.1-10 ng/ml) caused a significant dilation of the isolated lymph vessels with intact endothelium, which were precontracted with U46,619. The 10 ng/ml VEGF-induced dilation was significantly reduced by 3 x 10(-5) M N(omega)-nitro-L-arginine methyl ester (L-NAME). The action of L-NAME was inhibited by the simultaneous application of 10(-3) M L-arginine. Mechanical rubbing of the endothelium also caused significant inhibition of the VEGF-induced dilation. The findings suggest that VEGF(165) may activate the receptor-related tyrosine kinase and cause the release of Ca(2+) from the inositol 1,4, 5-triphosphate-sensitive intracellular Ca(2+) stores in LEC. VEGF(165) also produces endothelium-dependent nitric oxide-mediated dilation of the precontracted isolated lymph vessels.

WECHE: a novel hematopoietic regulatory factor.

Previously, we described AGM-derived endothelial cell lines that either inhibited or permitted the development of erythroid or B cells. We utilized a differential gene expression method to isolate a chemokine, termed WECHE, from one of these cell lines. WECHE inhibited the formation of erythroid cells but had no effect on either myeloid or B cell formation. WECHE repressed BFU-E development from either mouse fetal liver or bone marrow progenitor cells but had no effect on colony formation induced by IL-3 or IL-7. WECHE reduced HPP-CFC production from fetal liver-derived stem cells. WECHE hindered the growth of yolk sac-derived endothelial cells. WECHE was also chemotactic for bone marrow cells. Thus, WECHE is a novel chemokine that regulates hematopoietic differentiation.

Accessing endoscopic images for remote conference and diagnosis using WWW server with a secure socket layer.

Out of 137 hospitals in Niigata prefecture, 13 are connected to the Internet. The number of private clinics using the Internet has also increased. It is thought that an endoscopic information exchange over the Internet connection is useful for the cooperation between hospitals, clinics and organizations in related fields. A conventional World Wide Web (WWW) server of endoscopic images has been built around a compressed JPEG (Joint Panel Expert Group) format, for remote conferences and diagnoses. It has been made secure using a proxy server, a firewall server and a Secure Socket Layer (SSL). It is easy to access the server and view these endoscopic images using the usual homepage browsing method. Ordinarily, gastroenterologists would diagnose lesions with endoscopy in most cases. It is suggested that this conventional WWW server will be effective for remote conferences in some cases, even consultation will be possible.

The presence and role of the dopamine DA-2 receptor in the human decidua.

OBJECTIVES: Our objectives were to identify the presence of the dopamine DA-2 receptor in human decidua and to study its function in human parturition. METHODS: Human term decidual tissues were obtained during vaginal delivery and then homogenized. The P3 fraction was prepared for a radiolabeled receptor assay with [3H] spiperone as the ligand. Human decidual tissues obtained at cesarean section before the onset of labor were incubated in Krebs-Ringer buffer at 37 degrees C for 30 minutes in the presence of dopamine with or without (-)-sulpiride. The level of prostaglandin (PG) F in the medium was measured with a RIA kit. Differences were assessed with the Wilcoxon non-parametric test. RESULTS: Scatchard analysis showed a single class of binding sites having an equilibrium dissociation constant (Kd) of 2.25 +/- 0.59 nM (mean +/- SD) and a maximum binding capacity (Bmax) value of 166.5 +/- 77.7 fmol/mg protein (n = 3). Dopamine significantly increased the production of PGF. This stimulatory effect of dopamine was suppressed by (-)-sulpiride (p < 0.05; n = 7). CONCLUSION: The DA-2 receptor was demonstrated in the human decidua. Dopamine can stimulate PGF production via this receptor.

Commitment and differentiation of osteoclast precursor cells by the sequential expression of c-Fms and receptor activator of nuclear factor kappaB (RANK) receptors.

Osteoclasts are terminally differentiated cells derived from hematopoietic stem cells. However, how their precursor cells diverge from macrophagic lineages is not known. We have identified early and late stages of osteoclastogenesis, in which precursor cells sequentially express c-Fms followed by receptor activator of nuclear factor kappaB (RANK), and have demonstrated that RANK expression in early-stage of precursor cells (c-Fms(+)RANK(-)) was stimulated by macrophage colony-stimulating factor (M-CSF). Although M-CSF and RANKL (ligand) induced commitment of late-stage precursor cells (c-Fms(+)RANK(+)) into osteoclasts, even late-stage precursors have the potential to differentiate into macrophages without RANKL. Pretreatment of precursors with M-CSF and delayed addition of RANKL showed that timing of RANK expression and subsequent binding of RANKL are critical for osteoclastogenesis. Thus, the RANK-RANKL system determines the osteoclast differentiation of bipotential precursors in the default pathway of macrophagic differentiation.

[Home hospice care at a clinic].

For the people who want to stay at home until their last day, the primary doctor and clinic where they were diagnosed will be the most reliable supports. We have been operating a 19 bed clinic since 1996. In these three years, we have established what we call a "combination palliative care system." A team composed of two doctors, 13 nurses, 3 care aids, a social worker, and a counselor provides home care services as well as outpatient and inpatient care. From April, 1998 to March, 1999, 59 patients died of cancer. Among them, 25 patients died at home. Their primary cancers were lung (7), colon (3), pancreatic (2), breast (2), ovarian (2), brain (1), stomach (1), hepatoma (1), neck (1) and others. First of all, sufficient consultation with patients and family makes this care successful. Through this, the patient can choose his style of care. The whole staff is involved in this care in turn, so that all of us become acquainted with each patient. Home care includes: 1) medical and nursing service available 24 hours a day, 2) activation of social resources for the support of the patient user, 3) constructive cooperation with relevant institutions, 4) relieving the patient's physical and mental suffering, 5) aroma therapy, oil massage, hair cuts and music therapy, and 6) support by volunteers. In this way, as a neighborhood clinic, the combination palliative care system is valuable.

[Home palliative care of brain tumor patients from our clinic].

This study investigated whether or not home palliative care of brain tumor patients was possible and various ideas for accomplishing this, over the 2 1/2 years since our clinic was opened. There were 22 patients, aged 42-72 years, comprising 7 cases of glioblastoma, 14 cases of metastatic brain tumor, and 1 case of unknown origin. The control of intracranial hypertension and attacks of convulsion was possible with oral medication or suppositories, but not injection. The prognosis of cancer patients which changed home care showed no remarkable changes, and the home care rate was 53%. Malignant brain tumor patients were able to enjoy time with their families at home during the terminal stage, and die with dignity.