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X-ray free-electron lasers (FELs) enable crystallographic data collection using extremely bright femtosecond pulses from microscopic crystals beyond the limitations of conventional radiation damage. This diffraction-before-destruction approach requires a new crystal for each FEL shot and, since the crystals cannot be rotated during the X-ray pulse, data collection requires averaging over many different crystals and a Monte Carlo integration of the diffraction intensities, making the accurate determination of structure factors challenging. To investigate whether sufficient accuracy can be attained for the measurement of anomalous signal, a large data set was collected from lysozyme microcrystals at the newly established `multi-purpose spectroscopy/imaging instrument' of the SPring-8 Ångstrom Compact Free-Electron Laser (SACLA) at RIKEN Harima. Anomalous difference density maps calculated from these data demonstrate that serial femtosecond crystallography using a free-electron laser is sufficiently accurate to measure even the very weak anomalous signal of naturally occurring S atoms in a protein at a photon energy of 7.3 keV.

We have investigated multiphoton multiple ionization dynamics of xenon atoms using a new x-ray free-electron laser facility, SPring-8 Angstrom Compact free electron LAser (SACLA) in Japan, and identified that Xe(n+) with n up to 26 is produced at a photon energy of 5.5 keV. The observed high charge states (n≥24) are produced via five-photon absorption, evidencing the occurrence of multiphoton absorption involving deep inner shells. A newly developed theoretical model, which shows good agreement with the experiment, elucidates the complex pathways of sequential electronic decay cascades accessible in heavy atoms. The present study of heavy-atom ionization dynamics in high-intensity hard-x-ray pulses makes a step forward towards molecular structure determination with x-ray free-electron lasers.

We report a Bragg beam splitter developed for utilization of hard x-ray free-electron lasers. The splitter is based on an ultrathin silicon crystal operating in the symmetric Bragg geometry to provide high reflectivity and transmissivity simultaneously. We fabricated frame-shaped Si(511) and (110) crystals with thicknesses below 10 μm by a reactive dry etching method using atmospheric-pressure plasma. The thickness variation over an illuminated area is less than 300 nm peak-to-valley. High crystalline perfection was verified by topographic and diffractometric measurements. The crystal thickness was evaluated from the period of the Pendellösung beats measured with a highly monochromatic and collimated x-ray probe. The crystals provide two replica pulses with uniform wavefront [(<1/50)λ] and low spatial intensity variation (<5%). These Bragg beam splitters will play an important role in innovating XFEL applications.

LiF crystal and film detectors were used to measure the far-field fluence profile of a self-amplified spontaneous-emission free-electron laser beam and diffraction imaging with high spatial resolution. In these measurements the photoluminescence (PL) response of LiF crystal and film was compared over a wide range of soft x-ray fluences. It was found that the soft x-ray fluence dependences of LiF crystal and film differ. At low fluence, the LiF crystal shows higher PL response compared to LiF film, while this comparison is the opposite at higher fluence. Accurate measurement of LiF crystal and film PL response is important for precise characterization of the spatial, spectral, and coherence features of x-ray beams across the full profile and in localized areas. For such measurements, crucial LiF detector attributes are high spatial resolution and high dynamic range.

Ultrabrilliant, femtosecond X-ray pulses from X-ray free-electron lasers (XFELs) have promoted the investigation of exotic interactions between intense X-rays and matters, and the observation of minute targets with high spatio-temporal resolution. Although a single X-ray beam has been utilized for these experiments, the use of multiple beams with flexible and optimum beam parameters should drastically enhance the capability and potentiality of XFELs. Here we show a new light source of a two-colour double-pulse (TCDP) XFEL in hard X-rays using variable-gap undulators, which realizes a large and flexible wavelength separation of more than 30% with an ultraprecisely controlled time interval in the attosecond regime. Together with sub-10-fs pulse duration and multi-gigawatt peak powers, the TCDP scheme enables us to elucidate X-ray-induced ultrafast transitions of electronic states and structures, which will significantly contribute to the advancement of ultrafast chemistry, plasma and astronomical physics, and quantum X-ray optics.

We determined the pulse duration of x-ray free electron laser light at 10 keV using highly resolved single-shot spectra, combined with an x-ray free electron laser simulation. Spectral profiles, which were measured with a spectrometer composed of an ultraprecisely figured elliptical mirror and an analyzer flat crystal of silicon (555), changed markedly when we varied the compression strength of the electron bunch. The analysis showed that the pulse durations were reduced from 31 to 4.5 fs for the strongest compression condition. The method, which is readily applicable to evaluate shorter pulse durations, provides a firm basis for the development of femtosecond to attosecond sciences in the x-ray region.

We have developed a single-shot intensity-measurement system using a silicon positive-intrinsic-negative (PIN) photodiode for x-ray pulses from an x-ray free electron laser. A wide dynamic range (10(3)-10(11) photons/pulse) and long distance signal transmission (>100 m) were required for this measurement system. For this purpose, we developed charge-sensitive and shaping amplifiers, which can process charge pulses with a wide dynamic range and variable durations (ns-μs) and charge levels (pC-μC). Output signals from the amplifiers were transmitted to a data acquisition system through a long cable in the form of a differential signal. The x-ray pulse intensities were calculated from the peak values of the signals by a waveform fitting procedure. This system can measure 10(3)-10(9) photons/pulse of ~10 keV x-rays by direct irradiation of a silicon PIN photodiode, and from 10(7)-10(11) photons/pulse by detecting the x-rays scattered by a diamond film using the silicon PIN photodiode. This system gives a relative accuracy of ~10(-3) with a proper gain setting of the amplifiers for each measurement. Using this system, we succeeded in detecting weak light at the developmental phase of the light source, as well as intense light during lasing of the x-ray free electron laser., (© 2012 American Institute of Physics)

Optical features of point defects photoluminescence in LiF crystals, irradiated by soft X-ray pulses of the Free Electron Laser with wavelengths of 17.2 - 61.5 nm, were measured. We found that peak of photoluminescence spectra lies near of 530 nm and are associated with emission of F3+ centers. Our results suggest that redistribution of photoluminescence peak intensity from the red to the green part of the spectra is associated with a shortening of the applied laser pulses down to pico - or femtosecond durations. Dependence of peak intensity of photoluminescence spectra from the soft X-ray irradiation fluence was measured and the absence of quenching phenomena, even at relatively high fluencies was found, which is very important for wide applications of LiF crystal X-ray imaging detectors.

Nonlinear, three-photon double excitation of He in intense extreme ultraviolet free-electron laser fields (∼24.1  eV, ∼5  TW/cm2) is presented. Resonances to the doubly excited states converging to the He+ N=3 level are revealed by the shot-by-shot photoelectron spectroscopy and identified by theoretical calculations based on the time-dependent Schrödinger equation for the two-electron atom under a laser field. It is shown that the three-photon double excitation is enhanced by intermediate Rydberg states below the first ionization threshold, giving a greater contribution to the photoionization yields than the two-photon process by more than 1 order of magnitude.

We have observed and characterized 501.6 nm collective spontaneous emission (superfluorescence) following 1s(2) → 1s3p excitation of helium atoms by 53.7 nm free-electron laser radiation. Emitted pulse energies of up to 100 nJ are observed, corresponding to a photon number conversion efficiency of up to 10%. We observe the peak intensity to scale as ρ(2) and the emitted pulse width and delay to scale as ρ(-1), where ρ is the atom number density. Emitted pulses as short as 1 ps are observed, which corresponds to a rate around 75,000 times faster than the spontaneous 1s3p → 1s2s decay rate. To our knowledge, this is the first observation of superfluorescence following pumping in the extreme ultraviolet wavelength region, and extension of the technique to the generation of extreme ultraviolet and x-ray superfluorescence pulses should be straightforward by using suitable atomic systems and pump wavelengths.

As a novel approach to distinguish skeletal myogenic cell populations, basal lamina (BL) formation of myogenic cells was examined in the mouse compensatory enlarged plantaris muscles in vivo and in fiber-bundle cultures in vitro. MyoD(+) myogenic cells located inside the regenerative muscle fiber BL were laminin(-) but interstitial MyoD(+) cells were laminin(+). This was also confirmed by electron microscopy as structural BL formation. Similar trends were observed in the fiber-bundle cultures including satellite cells and interstitial myogenic cells and laminin(+) myogenic cells predominantly showed non-adhesive (non-Ad) behavior with Pax7(-), whereas laminin(-) cells were adhesive (Ad) with Pax7(+). Moreover, non-Ad/laminin(+) and Ad/laminin(-) myotubes were also observed and the former type showed spontaneous contractions, while the latter type did not. The origin and hierarchy of Ad/Pax7(+)/laminin(-) and non-Ad/Pax7(-)/laminin(+) myogenic cells were also examined using skeletal muscle interstitium-derived CD34(+)/45(-) (Sk-34) and CD34(-)/45(-) (Sk-DN) multipotent stem cells, which were composed of non-committed myogenic cells with a few (<1%) Pax7(+) cells in the Sk-DN cells at fresh isolation. Both cell types were separated by Ad/non-Ad capacity in repetitive culture. As expected, both Ad/Pax7(+)/laminin(-) and non-Ad/Pax7(-)/laminin(+) myogenic cells consistently appeared in the Ad and non-Ad cell culture. However, Ad/Pax7(+)/laminin(-) cells were repeatedly detected in the non-Ad cell culture, while the opposite phenomenon did not occur. This indicates that the source of non-Ad/ Pax7(-)/laminin(+) myogenic cells was present in the Sk-34 and Sk-DN stem cells and they were able to produce Ad/ Pax7(+)/ laminin(-) myogenic cells during myogenesis as primary myoblasts and situated hierarchically upstream of the latter cells.

We have developed an x-ray beam-position monitor for detecting the radiation properties of an x-ray free electron laser (FEL). It is composed of four PIN photodiodes that detect backscattered x-rays from a semitransparent diamond film placed in the beam path. The signal intensities from the photodiodes are used to compute the beam intensity and position. A proof-of-principle experiment at a synchrotron light source revealed that the error in the beam position is reduced to below 7 μm by using a nanocrystal diamond film prepared by plasma-enhanced chemical vapor deposition. Owing to high dose tolerance and transparency of the diamond film, the monitor is suitable for routine diagnostics of extremely intense x-ray pulses from the FEL.

BACKGROUND.: Postoperative neurogenic bladder dysfunction is a major complication of radical hysterectomy for cervical cancer and is mainly caused by unavoidable damage to the bladder branch of the pelvic plexus (BBPP) associated with colateral blood vessels. Thus, we attempted to reconstitute disrupted BBPP and blood vessels using skeletal muscle-derived multipotent stem cells that show synchronized reconstitution capacity of vascular, muscular, and peripheral nervous systems. METHODS.: Under pentobarbital anesthesia, intravesical pressure by electrical stimulation of BBPP was measured as bladder function. The distal portion of BBPP with blood vessels was then cut unilaterally (experimental neurogenic bladder model). Measurements were performed before, immediately after, and at 4 weeks after transplantation as functional recovery. Stem cells were obtained from the right soleus and gastrocnemius muscles after enzymatic digestion and cell sorting as CD34/45 (Sk-34) and CD34/45 (Sk-DN). Suspended cells were autografted around the damaged region, whereas medium alone and CD45 cells were transplanted as control groups. To determine the morphological contribution of the transplanted cells, stem cells obtained from green fluorescent protein transgenic mouse muscles were transplanted into a nude rat model and were examined by immunohistochemistry and immunoelectron microscopy. RESULTS.: At 4 weeks after surgery, the transplantation group showed significantly higher functional recovery ( approximately 80%) than the two controls ( approximately 28% and 24%). The transplanted cells showed an incorporation into the damaged peripheral nerves and blood vessels after differentiation into Schwann cells, perineurial cells, vascular smooth muscle cells, pericytes, and fibroblasts around the bladder. CONCLUSION.: Transplantation of multipotent Sk-34 and Sk-DN cells is potentially useful for the reconstitution of damaged BBPP.

The differentiation and/or therapeutic potential of skeletal muscle-derived stem cells for cardiac infarction have been studied extensively for use in cellular cardiomyoplasty, as injured cardiomyocytes exhibit limited regenerative capacity. We previously reported cardio-myogenic differentiation of skeletal muscle-derived CD34+/45(-) (Sk-34) stem cells after therapeutic transplantation. However, the clonal differentiation potential of these cells remains unknown. Here, we show that skeletal muscle-derived CD34(-)/45(-) (Sk-DN) stem cells, which are situated upstream of Sk-34 cells in the same lineage, exhibit clonal differentiation into cardiomyocytes after single cell-derived single-sphere implantation into myocardium. Sk-DN cells were enzymatically isolated from green fluorescent protein (GFP) transgenic mice and purified by flow cytometry, and were then clonally cultured in collagen-based medium with bFGF and EGF after clonal cell sorting. Single cell-derived single-sphere colonies of Sk-DN cells were directly implanted into the wild-type mouse myocardium. At 4 weeks after implantation, donor cells exhibited typical cardiomyocyte structure with the formation of gap-junctions between donor and recipient cells. Expression of specific mRNAs for cardiomyocytes, such as cardiac actin and GATA-4, Nkx2-5, Isl-1, Mef2, and Hand2, were also seen in clonal cell cultures of Sk-DN cells. Cell fusion-independent differentiation was also confirmed by bulk cell transplantation using Cre- and loxP (enhanced GFP)-mice. We conclude that Sk-DN cells can give rise to cardiac muscle cells clonally, and that skeletal muscle includes a practical cell source for cellular cardiomyoplasty.

Cellular responses in the compensatory hypertrophied (plantaris) muscle induced by surgical ablation of synergistic muscles (soleus and gastrocnemius) were determined during 10-week anabolic androgenic steroid (AAS) treatment. Adult Wistar male rats were divided randomly into the Control and Steroid groups, and contralateral surgery was performed. Nandrolone decanoate was administered to the Steroid group. [3H]thymidine and [14C]leucine labeling were used to determine the serial changes in cellular mitotic activity and amino acid uptake. Myogenic cells and cellular responses in blood vessels and nerve fibers were analyzed by immunohistochemistry. Significantly lower cellular mitotic activity associated with lower volume of muscle fiber necrosis was observed in the Steroid group during the first week. However, amino acid uptake and final muscle wet weight gain did not differ between the groups. Marked activation/proliferation of muscular, vascular, and peripheral nerve-related cells was seen with the inflammatory responses in both groups. However, this activation was dependent on the volume of muscle fiber damage and was not preferentially accelerated by AAS loading. These results indicated that AAS loading significantly diminished muscle fiber damages, but they did not accelerate final muscle wet weight gain and activation of myogenic, vascular, and peripheral nerve related cells in the compensatory enlarged muscles.

Tissue inflammation and multiple cellular responses in the compensatory enlarged plantaris (OP Plt) muscle induced by surgical ablation of synergistic muscles (soleus and gastrocnemius) were followed over 10 weeks after surgery. Contralateral surgery was performed in adult Wistar male rats. Cellular responses in muscle fibers, blood vessels and nerve fibers were analyzed by immunohistochemistry and electron microscopy. Severe muscle fiber damage and disappearance of capillaries associated with apparent tissue edema were observed in the peripheral portion of OP Plt muscles during the first week, whereas central portions were relatively preserved. Marked cell activation/proliferation was also mainly observed in peripheral portions. Similarly, activated myogenic cells were seen not only inside but also outside of muscle fibers. The former were likely satellite cells and the latter may be interstitial myogenic cells. One week after surgery, small muscle fibers, small arteries and capillaries and several branched-muscle fibers were evident in the periphery, thus indicating new muscle fiber and blood vessel formation. Proliferating cells were also detected in the nerve bundles in the Schwann cell position. These results indicate that the compensatory stimulated/enlarged muscle is a suitable model for analyzing multiple physiological cellular responses in muscle-nerve-blood vessel units under continuous stretch stimulation.

Solvation structures of an excess proton in protonated methanol cluster ions, H(+)(CH(3)OH)(n) (n=5-8), were investigated by photodissociation spectroscopy in the middle infrared region (900-2300 cm(-1)) and by using density-functional theory. This work indicates that the excess proton is delocalized between two methanol molecules. Spectral features observed in the range 1400-1800 cm(-1) are attributed to vibrational modes involving collective motion of the shared proton and the two ligand molecules. At n=6-8, broad spectral features in the region above 1800 cm(-1) suggest coexistence of isomers in which the excess proton and a methanol molecule are tightly bound to form an ion core, CH(3)OH(2) (+).

The hierarchical relationship of skeletal muscle-derived multipotent stem cells sorted as CD34(+)/CD45(-) (Sk-34) and CD34(-)/CD45(-) (Sk-DN) cells, which have synchronized reconstitution capacities for blood vessels, peripheral nerves, and muscle fibers, was examined. Expression of Sca-1 and CD34 (typical state of freshly isolated Sk-34 cells) in Sk-DN cells was examined using in vitro culture and in vivo cell implantation. Sk-DN cells sequentially expressed Sca-1 and CD34 during cell culture showing self-maintenance and/or self-renewal-like behavior, and are thus considered hierarchically upstream of Sk-34 cells in the same lineage. Sk-34 and Sk-DN cells were further divided into small and large cell fractions by cell sorting. Immunocytochemistry using anti-Pax7 was performed at the time of isolation (before culture) and revealed that only 1% of cells in the large Sk-DN cell fraction were positive for Pax7, while Sk-34 cells and 99% of Sk-DN cells were negative for Pax7. Therefore, putative satellite cells were possibly present in the large Sk-DN cell fraction. However, serial analysis of Pax7 expression by RT-PCR and immunocytochemistry for single and 2 to >40 clonally proliferated Sk-34 and Sk-DN cells revealed that both cell types expressed Pax7 after several asymmetric cellular divisions during clonal-cell culture. In addition, production of satellite cells was seen after muscle fiber formation following Sk-34 or Sk-DN cell transplantation into damaged muscle, and even in the nonmuscle tissue environment (beneath the renal capsule). Thus, Sk-DN cells are situated upstream of Sk-34 cells and both cells can produce Pax7+ cells (putative satellite cells) after cellular division.

Background: Postoperative damage of the urethral rhabdosphincter (URS) and neurovascular bundle (NVB) is a major operative complication of radical prostatectomy. It is generally recognized to be caused by unavoidable surgical damage to the muscle-nerve-blood vessel units around the urethra. We attempted to treat this damage using skeletal muscle-derived stem cells, which are able to reconstitute muscle-nerve-blood vessel units., Methods: Cells were enzymatically extracted and sorted by flow cytometry as CD34/45 (Sk-34) and CD34/45 (Sk-DN) cells from green fluorescent protein transgenic mice and rats. URS-NVB damage was induced by manually removing one-third of the total URS and unilateral invasion of NVB in wild-type Sprague-Dawley and node rats. Freshly isolated Sk-34, Sk-34+Sk-DN cells, and cultured Sk-DN cells were directly transplanted into the damaged portion., Results: At 4 and 12 weeks after transplantation, urethral pressure profile by electrical stimulation through the sacral surface (L6-S1) was evaluated as functional recovery. The recovery ratio in the control and transplanted groups was 37.6% and 72.9%, at 4 weeks, and 41.6% and 78.4% at 12 weeks, respectively (P<0.05). Immunohistochemical and immunoelectron microscopic analysis revealed that transplanted cells differentiated into numerous skeletal muscle fibers having neuromuscular junctions (innervation) and nerve bundle-related Schwann cells and perineurium, and blood vessel-related endothelial cells and pericyte around the urethra., Conclusions: Thus, we conclude that transplantation of skeletal muscle-derived multipotent Sk-34 and Sk-DN cells is potentially useful for the reconstitution of postoperative damage of URS and NVB after radical prostatectomy.

Background: Cellular cardiomyoplasty for myocardial infarction has been developed using various cell types. However, complete differentiation and/or trans-differentiation into cardiomyocytes have never occurred in these transplant studies, whereas functional contributions were reported., Methods and Results: Skeletal muscle interstitium-derived CD34(+)/CD45(-) (Sk-34) cells were purified from green fluorescent protein transgenic mice by flowcytometory. Cardiac differentiation of Sk-34 cells was examined by in vitro clonal culture and co-culture with embryonic cardiomyocytes, and in vivo transplantation into a nude rat myocardial infarction (MI) model (left ventricle). Lower relative expression of cardiomyogenic transcription factors, such as GATA-4, Nkx2-5, Isl-1, Mef2 and Hand2, was seen in clonal cell culture. However, vigorous expression of these factors was seen on co-culture with embryonic cardiomyocytes, together with formation of gap-junctions and synchronous contraction following sphere-like colony formation. At 4 weeks after transplantation of freshly isolated Sk-34 cells, donor cells exhibited typical cardiomyocyte structure with formation of gap-junctions, as well as intercalated discs and desmosomes, between donor and recipient and/or donor and donor cells. Fluorescence in situ hybridization (FISH) analysis detecting the rat and mouse genomic DNA and immunoelectron microscopy using anti-GFP revealed donor-derived cells. Transplanted Sk-34 cells were incorporated into infarcted portions of recipient muscles and contributed to cardiac reconstitution. Significant improvement in left ventricular function, as evaluated by transthoracic echocardiography and micro-tip conductance catheter, was also observed., Conclusions and Significance: Skeletal muscle-derived multipotent Sk-34 cells that can give rise to skeletal and smooth muscle cells as reported previously, also give rise to cardiac muscle cells as multi-myogenic stem cells, and thus are a potential source for practical cellular cardiomyoplasty.

We have already confirmed that cell sheet transplantation can improve damaged heart function via continuous cytokine secretion. In this study, we hypothesized that cytokine-secreting cell sheets co-cultured with an endothelial cell source may be more effective for repairing ischemic myocardium. Confluent rat fibroblasts cultured on temperature-responsive culture dishes were harvested as contiguous cell sheets by temperature reduction. Green fluorescent protein (GFP)-positive endothelial progenitor cells (EPCs) were seeded on fibroblast sheets to create co-cultured cell sheets, and sandwich-like constructs were engineered by stacking of the co-cultured cell sheets. These constructs were transplanted into rat myocardial infarction models. Cardiac function and histology were assessed in four groups: the sham operation (C) group, the isolated EPC injection (E) group, the transplantation of triple-layer fibroblast sheets (F) group, and the transplantation of triple-layer sandwich-like constructs (E + F) group. Echocardiography showed significant improvement of the fractional shortening in the E + F group in comparison with the C group (0.25 +/- 0.05 vs. 0.16 +/- 0.02). On histological examination, significantly less connective tissue formation was observed in the E, F, and E + F groups when compared to the C group (C, E, F, and E + F groups: 53 +/- 2%, 41 +/- 4%, 40 +/- 4%, and 32 +/- 7%, respectively). Additionally, increased blood vessel formation was detected in the E, F, and E + F groups compared with the C group (C, E, F, and E + F groups: 1.9% +/- 0.6%, 6.7% +/- 0.6%, 7.8% +/- 0.9%, and 10.2% +/- 2.4%, respectively). Furthermore, GFP-staining demonstrated that the newly formed blood vessels were composed of the co-cultured EPCs. Transplantation of cell sheets co-cultured with an endothelial cell source may be a new therapeutic strategy for myocardial tissue regeneration.

In order to establish the practical isolation and usage of skeletal muscle-derived stem cells (MDSCs), we determined reconstitution capacity of CD34(-)/CD45(-) (Sk-DN) cells as a candidate somatic stem cell source for transplantation. Sk-DN cells were enzymatically isolated from GFP transgenic mice (C57/BL6N) skeletal muscle and sorted using fluorescence activated cell sorting (FACS), and expanded by collagen gel-based cell culture with bFGF and EGF. The number of Sk-DN cells was small after sorting (2-8 x 10(4)); however, the number increased 10-20 fold (2-16 x 10(5)) after 6 days of expansion culture, and the cells maintained immature state and multipotency, expressing mRNAs for mesodermal and ectodermal cell lineages. Transplantation of expanded Sk-DN cells into the severe muscle damage model (C57/BL6N wild-type) resulted in the synchronized reconstitution of blood vessels, peripheral nerves and muscle fibers following significant recovery of total muscle mass (57%) and contractile function (55%), whereas the non-cell-transplanted control group showed around 20% recovery in both factors. These reconstitution capacities were supported by the intrinsic plasticity of Sk-DN cells that can differentiate into muscular (skeletal muscle), vascular (pericyte, endothelial cell and smooth muscle) and peripheral nerve (Schwann cells and perineurium) cell lineages that was revealed by transplantation to non-muscle tissue (beneath renal capsule) and fluorescence in situ hybridization (FISH) analysis.

The differentiation potential of skeletal muscle-derived stem cells (MDSCs) after in vitro culture and in vivo transplantation has been extensively studied. However, the clonal multipotency of MDSCs has yet to be fully determined. Here, we show that single skeletal muscle-derived CD34-/CD45- (skeletal muscle-derived double negative [Sk-DN]) cells exhibit clonal multipotency that can give rise to myogenic, vasculogenic, and neural cell lineages after in vivo single cell-derived single sphere implantation and in vitro clonal single cell culture. Muscles from green fluorescent protein (GFP) transgenic mice were enzymatically dissociated and sorted based on CD34 and CD45. Sk-DN cells were clone-sorted into a 96-well plate and were cultured in collagen-based medium with basic fibroblast growth factor and epidermal growth factor for 14 days. Individual colony-forming units (CFUs) were then transplanted directly into severely damaged muscle together with 1 x 10(5) competitive carrier Sk-DN cells obtained from wild-type mice muscle expanded for 5 days under the same culture conditions using 35-mm culture dishes. Four weeks after transplantation, implanted GFP+ cells demonstrated differentiation into endothelial, vascular smooth muscle, skeletal muscle, and neural cell (Schwann cell) lineages. This multipotency was also confirmed by expression of mRNA markers for myogenic (MyoD, myf5), neural (Musashi-1, Nestin, neural cell adhesion molecule-1, peripheral myelin protein-22, Nucleostemin), and vascular (alpha-smooth muscle actin, smoothelin, vascular endothelial-cadherin, tyrosine kinase-endothelial) stem cells by clonal (single-cell derived) single-sphere reverse transcription-polymerase chain reaction. Approximately 70% of clonal CFUs exhibited expression of all three cell lineages. These findings support the notion that Sk-DN cells are a useful tool for damaged muscle-related tissue reconstitution by synchronized vasculogenesis, myogenesis, and neurogenesis.

Infrared photodissociation action spectra of protonated ammonia cluster ions, NH(4) (+)(NH(3))(n) (n=5-8), were measured in the range of 1020-1210 cm(-1) by using a tunable infrared free electron laser. Analyses by the density functional theory (DFT) show that the spectral features observed can be assigned to the nu(2) vibrational mode of the NH(3) molecules in NH(4) (+)(NH(3))(n). Size dependence of the spectra supports structural models obtained by the DFT calculations, in which the NH(4) (+) ion is solvated by the four nearest-neighbor NH(3) molecules. For NH(4) (+)(NH(3))(5), the spectrum between 1000 and 1700 cm(-1) was measured. The nu(4) bands of the NH(3) molecules and the NH(4) (+) ion were found in the range of 1420-1700 cm(-1).

Objective: Tissue regeneration requires both growth factor and extracellular matrix such as collagen, serving as a scaffold for cell growth. We established FNCBD-VEGF121, consisting of the fibronectin collagen-binding domain (FNCBD) and vascular endothelial growth factor (VEGF) 121, and investigated its properties., Methods and Results: FNCBD-VEGF121 specifically bound to gelatin and type I, II, III, IV, and V collagen. This collagen-bound FNCBD-VEGF121 captured soluble VEGF receptor 2 (VEGFR-2)/Fc chimeric protein. Cell growth-promoting activity of FNCBD-VEGF121 was almost identical to that of VEGF121. The VEGF fusion protein significantly enhanced the expression of VEGFR-2 (71.6+/-0.8%) on endothelial progenitor cells (EPCs) derived from umbilical cord blood. Expectably, the collagen-bound VEGF fusion protein not only promoted the growth of endothelial cells (ECs) but also induced the expression of VEGFR-2 (63.7+/-0.8%) on non-adherent cells expanded from bone marrow CD34+ cells. Moreover, the VEGF fusion protein enhanced sprout formation of ECs in a matrigel model. In vivo experiments revealed that FNCBD-VEGF121 had local effects but not systemic effect on EPC mobilization., Conclusions: These results suggest that FNCBD-VEGF121 stably maintains an optimally high and local concentration of VEGF with collagen matrix and stimulates both ECs and EPCs in situ, supplying a vascular regeneration niche.

The photodissociation of manganese oxide cluster cations Mn(N)O+ (N = 2-5), into Mn(N-1)O+ (one-atom loss) and Mn(N-2)O+ (two-atom), was investigated in the photon-energy range of 1.08-2.76 eV. The bond-dissociation energies D0(Mn(N-1)O+...Mn) for N = 3, 4, and 5 were determined to be 1.84+/-0.03, 0.99+/-0.05, and 1.25+/-0.14 eV, respectively, from the threshold energies for the one- and two-atom losses. As Mn2O+ did not dissociate even at the highest photon energy used, the bond dissociation energy of Mn2O+, D0(Mn+...MnO), was obtained from a density-functional-theory calculation to be 3.04 eV. The present findings imply that the core ion Mn2O+ is bound weakly with the rest of the manganese atoms in Mn(N)O+.

The binding energies of manganese cluster ions Mn(N)+ (N = 5-7) were determined by the photodissociation experiments in the near-infrared and visible-photon-energy ranges. The bond dissociation energies of Mn(N)+, D0(Mn(N-1)+...Mn), were obtained to be 1.70+/-0.08, 1.04+/-0.10, and 1.46+/-0.11 eV, respectively, for N = 5, 6, and 7 from the threshold energies for the two-atom loss processes and the bond dissociation energies of Mn3(+) and Mn4(+) reported previously [A. Terasaki et al., J. Chem. Phys. 117, 7520 (2002)]. Correspondingly, binding energies per atom are obtained to be 0.99+/-0.03, 1.00+/-0.03, and 1.06+/-0.03 eV/at. for N = 5, 6, and 7, respectively. A gradual increase in the binding energy from N = 2 to N = 7 shows an increasing contribution of nonbonding 3d orbitals to the bonding via weak hybridization with valence 4s orbitals as the cluster size increases. These binding energies per atom are still much smaller than the bulk cohesive energy of manganese (2.92 eV/at.), and this finding indicates exceptionally weak metal-metal bonds in this size range.

The response of gingival fibroblasts cultured from humans with gingival fibromatosis to phenytoin (PHT) and nifedipine (NIF) was investigated. PHT and NIF induced proliferation, and increased the expression of immunoreactive endothelin-1 (ET-1). ET-1 (0.1 nm-1 microm) itself also induced proliferation in a concentration-dependent manner. The proliferation was inhibited by BQ-123 (ETA receptor antagonist; 1 microm) and TAK044 (ETA/ETB receptor antagonist; 1 microm), but not by BQ-788 (ETB receptor antagonist; 1 microm). The proliferation induced by PHT (0.25 microm) and NIF (0.25 microm) was inhibited by BQ-123 (1 microm). In addition, the results of Western blot analysis indicated the presence of ETA and ETB receptors in/on the fibroblasts. These findings suggest that PHT- and NIF-induced gingival proliferation may be mediated by endogenously generated ET-1, possibly via ETA receptors.

The photoelectron spectrum of the dichromium oxide cluster anion, Cr2O-, and the analysis by the density-functional theory revealed that the spins of the two Cr atoms in Cr2O- are ferromagnetically coupled, and that its total spin magnetic moment is as large as 9 mu(B). This ferromagnetic spin coupling is induced by oxidation; the mixing of Cr 3d with O 2p orbitals plays an important role in a spin coupling between the localized electrons at the two Cr sites bridged by the O atom. The present finding is in marked contrast to the pure chromium dimer, which is known to be antiferromagnetic due to the strong sextuple Cr-Cr bond.

The initial oxygen adsorption on the Si(111)7 x 7 surface was investigated by high-resolution x-ray absorption spectroscopy. Below 220 K, a molecular adsorption species is identified by distinctive absorption resonances due to the 1 pi(g) molecular orbitals. The molecular species is metastabilized to have a lifetime of 15-35 min at 135 K only with the presence of atomic adsorbates of more than 0. 1 ML (monolayer). It is thus clearly evidenced that the very initial adsorption is dissociative even at 100 K and the molecular species is not a precursor state. The molecular adsorption structures with the coadsorbed oxygen atoms are suggested.