Self-Guiding of Long-Wave Infrared Laser Pulses Mediated by Avalanche Ionization.

Nonlinear self-guided propagation of intense long-wave infrared (LWIR) laser pulses is of significant recent interest, as it promises high power transmission without beam breakup and multifilamentation. Central to self-guiding is the mechanism for the arrest of self-focusing collapse. Here, we show that discrete avalanche sites centered on submicron aerosols can arrest self-focusing, providing a new mechanism for self-guided propagation of moderate intensity LWIR pulses in outdoor environments. Our conclusions are supported by simulations of LWIR pulse propagation using an effective index approach that incorporates the time-resolved plasma dynamics of discrete avalanche breakdown sites.

Absolute Measurement of Laser Ionization Yield in Atmospheric Pressure Range Gases over 14 Decades.

Strong-field ionization is central to intense laser-matter interactions. However, standard ionization measurements have been limited to extremely low density gas samples, ignoring potential high density effects. Here, we measure strong-field ionization in atmospheric pressure range air, N_{2}, and Ar over 14 decades of absolute yield, using mid-IR picosecond avalanche multiplication of single electrons. Our results are consistent with theoretical rates for isolated atoms and molecules and quantify the ubiquitous presence of ultralow concentration gas contaminants that can significantly affect laser-gas interactions.

Adaptive control of laser-wakefield accelerators driven by mid-IR laser pulses.

There has been growing interest both in studying high intensity ultrafast laser plasma interactions with adaptive control systems as well as using long wavelength driver beams. We demonstrate the coherent control of the dynamics of laser-wakefield acceleration driven by ultrashort (∼ 100 fs) mid-infrared (∼ 3.9 µm) laser pulses. The critical density at this wavelength is 7.3 × 1019 cm-3, which is achievable with an ordinary gas target system. Interactions between mid-infrared laser pulses and such near-critical-density plasma may be beneficial due to much higher absorption of laser energy. In addition, the normalized vector potential of the laser field a0 increases with longer laser wavelength, lowering the required peak laser intensity to drive non-linear laser-wakefield acceleration. Here, MeV level, collimated electron beams with non-thermal, peaked energy spectra are generated. Optimization of electron beam qualities are realized through adaptive control of the laser wavefront. A genetic algorithm controlling a deformable mirror improves the electron total charge, energy spectra, beam pointing and stability at various plasma density profiles. Particle-in-cell simulations reveal that the optimal wavefront causes an earlier injection on the density up-ramp and thus higher energy gain as well as less filamentation during the interaction, which leads to the improvement in electron beam collimation and energy spectra.

Laser wakefield acceleration with mid-IR laser pulses.

We report on, to the best of our knowledge, the first results of laser plasma wakefield acceleration driven by ultrashort mid-infrared (IR) laser pulses (λ=3.9 µm, 100 fs, 0.25 TW), which enable near- and above-critical density interactions with moderate-density gas jets. Relativistic electron acceleration up to ∼12 MeV occurs when the jet width exceeds the threshold scale length for relativistic self-focusing. We present scaling trends in the accelerated beam profiles, charge, and spectra, which are supported by particle-in-cell simulations and time-resolved images of the interaction. For similarly scaled conditions, we observe significant increases in the accelerated charge, compared to previous experiments with near-infrared (λ=800 nm) pulses.

MeV electron acceleration at 1 kHz with <10 mJ laser pulses: erratum.

In this erratum the funding section of Opt. Lett.42, 215 (2017)OPLEDP0146-959210.1364/OL.42.000215 has been updated.

MeV electron acceleration at 1 kHz with <10 mJ laser pulses.

We demonstrate laser-driven acceleration of electrons to MeV-scale energies at 1 kHz repetition rate using <10 mJ pulses focused on near-critical density He and H2 gas jets. Using the H2 gas jet, electron acceleration to ∼0.5 MeV in ∼10 fC bunches was observed with laser pulse energy as low as 1.3 mJ. Increasing the pulse energy to 10 mJ, we measure ∼1 pC charge bunches with >1 MeV energy for both He and H2 gas jets.

Fate of amnion-derived stem cells transplanted to the fetal rat brain: migration, survival and differentiation.

We have recently characterized a stem cell population isolated from the rodent amniotic membrane termed amnion-derived stem cells (ADSCs). In vitro ADSCs differentiate into cell types representing all three embryonic layers, including neural cells. In this study we evaluated the neuroectodermal potential of ADSCs in vivo after in utero transplantation into the developing rat brain. A clonal line of green fluorescent protein-expressing ADSCs were infused into the telencephalic ventricles of the developing embryonic day 15.5 rat brain. At E17.5 donor cells existed primarily as spheres in the ventricles with subsets fused to the ventricular walls, suggesting a mode of entry into the brain parenchyma. By E21.5 green fluorescent protein (GFP) ADSCs migrated to a number of brain regions. Examination at postnatal time points revealed that donor ADSCs expressed vimentin and nestin. Subsets of transplanted ADSCs attained neuronal morphologies, although there was no immunohistochemical evidence of neural or glial differentiation. Some donor cells migrated around blood vessels and differentiated into putative endothelial cells. Donor ADSCs transplanted in utero were present in recipients into adulthood with no evidence of immunological rejection or tumour formation. Long-term survival may suggest utility in the treatment of disorders where differentiation to a neural cell type is not required for clinical benefit.

Long-term cryopreserved amniocytes retain proliferative capacity and differentiate to ectodermal and mesodermal derivatives in vitro.

Putative stem cells have recently been isolated from several extra-embryonic tissues, including Wharton's Jelly and umbilical cord blood. Relevant studies have focused on primary cultures established from freshly isolated tissues. In this report, we examine the plasticity of 472 cells, a cryopreserved human amniocyte cell line originally isolated in 1974. Under conditions conducive for proliferation, the amniocytes displayed fibroblast-like morphologies and expressed Oct4 and Rex1, genes associated with pluripotency. Perhaps indicative of inherent plasticity, 472 cells simultaneously expressed ectodermal beta-III-tubulin and mesodermal fibronectin. When cultured under conditions that promote neural differentiation, the cells adopted neuronal morphologies and expressed neuronal genes, including Gap-43, NF-M, tau, and synaptophysin. Exposure to culture conditions that encourage osteogenic differentiation resulted in increased expression of alkaline phosphatase (ALP) and the deposition of mineralized matrix, established markers of bone cell differentiation. In sum, this population of human amniocytes appears to be multipotent, capable of in vitro differentiation to ectodermal and mesodermal cell types. Retention of this plasticity through decades of cryopreservation suggests that amniocytes might be candidates for future cell-based therapies.

Characterization of reconstituted Fo from wild-type Escherichia coli and identification of two other fluxes co-purifying with Fo.

We purified the ATPase Fo sector from a nonoverexpressing strain of Escherichia coli, reconstituted it into lipid vesicles made of either asolectin or two different mixtures of purified lipids, and measured proton flux through the reconstituted proton channel. We measured single-channel conductances and found that Fo activity depends on both lipids and reconstitution methods. In asolectin vesicles, Fo has a single-channel conductance of about 0.2 fS. Additionally, the relatively impure Fo prepared from cells carrying single-copy ATPase genes allowed us to observe two other fluxes, a nonselective cation leak (C(L)) and a slow H+ flux (Hs). Unlike the Fo flux, these fluxes could not be blocked by the Fo inhibitor DCCD. The C, reduces the total apparent trapped volume inside vesicles and therefore must equilibrate both H+ and K+ in the vesicles that contain it. When reconstituted into bilayers, these Fo preparations displayed a 120 pS cation channel with characteristics consistent with C(L) flux. The Hs conducts only H+ but at a slower rate than the Fo. We were therefore able to: 1) quantitate the single-channel conductance of the Fo, 2) demonstrate that our Fo purification method co-purified other membrane proteins that have ion-conduction properties, and 3) show that certain lipids are necessary for functional reconstitution of Fo.

The t-SNARE syntaxin is sufficient for spontaneous fusion of synaptic vesicles to planar membranes.

Vesicular trafficking and exocytosis are directed by the complementary interaction of membrane proteins that together form the SNARE complex. This complex is composed of proteins in the vesicle membrane (v-SNAREs) that intertwine with proteins of the target membrane (t-SNAREs). Here we show that modified synaptic vesicles (mSV), containing v-SNAREs, spontaneously fuse to planar membranes containing the t-SNARE, syntaxin 1A. Fusion was Ca(2+)-independent and did not occur with vesicles lacking v-SNAREs. Therefore, syntaxin alone forms a functional fusion complex with v-SNAREs. Our functional fusion assay uses synaptic vesicles that are modified, so each fusion event results in an observable transient current. The mSV do not fuse with protein-free membranes. Additionally, artificial vesicles lacking v-SNAREs do not fuse with membranes containing syntaxin. This technique can be adapted to measure fusion in other SNARE systems and should enable the identification of proteins critical to vesicle-membrane fusion. This will further our understanding of exocytosis and may improve targeting and delivery of therapeutic agents packaged in vesicles.

Adult rat and human bone marrow stromal cells differentiate into neurons.

Bone marrow stromal cells exhibit multiple traits of a stem cell population. They can be greatly expanded in vitro and induced to differentiate into multiple mesenchymal cell types. However, differentiation to non-mesenchymal fates has not been demonstrated. Here, adult rat stromal cells were expanded as undifferentiated cells in culture for more than 20 passages, indicating their proliferative capacity. A simple treatment protocol induced the stromal cells to exhibit a neuronal phenotype, expressing neuron-specific enolase, NeuN, neurofilament-M, and tau. With an optimal differentiation protocol, almost 80% of the cells expressed NSE and NF-M. The refractile cell bodies extended long processes terminating in typical growth cones and filopodia. The differentiating cells expressed nestin, characteristic of neuronal precursor stem cells, at 5 hr, but the trait was undetectable at 6 days. In contrast, expression of trkA, the nerve growth factor receptor, persisted from 5 hr through 6 days. Clonal cell lines, established from single cells, proliferated, yielding both undifferentiated and neuronal cells. Human marrow stromal cells subjected to this protocol also differentiated into neurons. Consequently, adult marrow stromal cells can be induced to overcome their mesenchymal commitment and may constitute an abundant and accessible cellular reservoir for the treatment of a variety of neurologic diseases.

Endothelin-1 in hypertension in the baroreflex-intact SHR: a role independent from vasopressin release.

This study sought to identify whether central endothelin (ET) receptor activation contributes to the elevated pressure in spontaneously hypertensive rats (SHR) and whether an ET-stimulated vasopressin (AVP) release mediates the increased pressure. In Wistar Kyoto (WKY) rats, intracerebroventricular ET-1 induced a dose-dependent pressor response that was shifted rightward in SHR. ET(A) antagonism decreased mean arterial pressure in baroreflex-intact SHR (P<0.01), consistent with inhibition of endogenous ET-1, and blocked the pressor response to exogenous ET-1 in both strains. ET-1 increased AVP only after sinoaortic denervation (P<0.05). Contrary to WKY, sinoaortic denervation was required to elicit a significant pressor response with 5 pmol ET-1 in SHR. Sinoaortic denervation permitted ET-1 to increase AVP in both strains, and peripheral V(1) blockade decreased pressure in denervated but not intact rats. After nitroprusside normalized pressure in SHR, the pressor and AVP secretory responses paralleled those in WKY. Thus endogenous ET(A) receptor mechanisms contribute to hypertension, independent of AVP, in baroreflex-intact SHR. Although blunted in the hypertensive state, the arterial baroreflex buffers the ET-1-induced pressor and AVP secretory responses in both strains.

Desensitization and resensitization of lutropin receptors expressed in transfected Y-1 adrenal cells.

Stimulation of gonadal cells by lutropins such as human chorionic gonadotropin (hCG) is often transient and followed by down-regulation and/or desensitization of lutropin receptors (LHR). Here we describe desensitization/resensitization of LHR in Y-1 adrenal cell lines (termed Y-1L) expressing a rat cDNA lacking most 5' and 3' LHR untranslated regions under the control of a metallothionein promoter. Using a simple morphological assay in which stimulated cells are round and unstimulated cells are flat, we identified clones that rounded and remained round and others that became insensitive to lutropin stimulation and reverted to their flat appearance within 2-4 h. Flattened cells were insensitive to further hormonal stimulation but rounded after treatments with cholera toxin, forskolin, or cyclic AMP, showing that loss of responsiveness was associated with an early step in signal transduction, not loss of rounding potential. Removing the lutropin stimulus for at least 90-120 min reversed hormone insensitivity, even in the presence of the protein synthesis inhibitor puromycin. The number of surface bound receptors did not change during a cycle of rounding/flattening and hCG bound to rounded or flattened cells was replaced equally by radioiodinated hCG during incubations at 4 degrees C. Thus, desensitization/resensitization of LHR in Y-1L cells occurred in the absence of new receptor synthesis, receptor degradation, or receptor recycling. These observations suggest that LHR desensitization/resensitization in Y-1L cells was closely coupled to receptor occupancy and that this cell line may be useful for identifying factors that modulate the activities of occupied receptors.

Building a bilayer model of the neuromuscular synapse.

Progress over the past 10 years has made it possible to construct a simple model of neurotransmitter release. Currently, some models use artificially formed vesicles to represent synaptic vesicles and a planar lipid bilayer as a presynaptic membrane. Fusion of vesicles with the bilayer is via channel proteins in the vesicle membrane and an osmotic gradient. In this paper; a framework is presented for the successful construction of a more complete model of synaptic transmission. This model includes real synaptic vesicles that fuse with a planar bilayer. The bilayer contains acetylcholine receptor (AChR) channels which function as autoreceptors in the membrane. Vesicle fusion is initiated following a Ca2+ flux through voltage-gated Ca2+ channels. Key steps in the plan are validated by mathematical modeling. Specifically, the probability that a reconstituted AChR channel opens following the release of ACh from a fusing vesicle, is calculated as a function of time, quantal content, and number of reconstituted AChRs. Experimentally obtainable parameters for construction of a working synapse are given. The inevitable construction of a full working model will mean that the minimal structures necessary for synaptic transmission are identified. This will open the door in determining regulatory and modulatory factors of transmitter release.

Nystatin/ergosterol method for reconstituting ion channels into planar lipid bilayers.

The nystatin-ergosterol (N/E) method is described and reviewed. Using this procedure, an experimenter can promote and detect fusion of vesicles with planar lipid bilayers. N/E fusion provides a straightforward mechanism to reconstitute any membrane protein into planar lipid bilayers. Once reconstituted, it is easy to determine the ion selectivity, transport rate, voltage dependence, and kinetics of any conductance caused by the membrane protein. Fusigenic N/E vesicles are made with a mixture of phospholipids, ergosterol, and nystatin. Vesicle size can be adjusted either with sonication or with polycarbonate filters. The best vesicles contain approximately 20 mol% ergosterol, are approximately 200 nm in diameter, and are in a solution containing approximately 50 micrograms/ml nystatin. Vesicle fusion requires an osmotic gradient and delivery of vesicles to the bilayer. Vesicle delivery is increased by (1) stirring of the chamber that contains vesicles, (2) larger bilayers, and (3) bilayers that are face-flush with the vesicle-containing solution. Because constant stirring is critical for delivery of vesicles to the bilayer, a system that allows simultaneous stirring and sensitive electrical measurements is desirable. The main strength of the bilayer technique has always been that the experimenter has control over the milieu of the membrane system. The N/E fusion technique adds to this strength by controlling fusion of vesicles to the bilayer, thus allowing the quantitative transfer of isolated proteins from vesicle to bilayer. The techniques and calculations necessary for successful quantitative reconstitution are given in detail.

Novel structure of the human GDNF gene.

Glial cell line-derived neurotrophic factor (GDNF) is the most potent known survival factor for substantia nigra neurons, which degenerate in Parkinson's disease, for spinal motoneurons, which die in Lou Gehrig's disease (ALS), and for Purkinje neurons, the critical outflow cells of the cerebellum. Moreover, targeted deletion of the GDNF gene results in renal dysgenesis and abnormal development of the enteric nervous system. GDNF mRNA is expressed in a complex temporospatial pattern in the central nervous system and the periphery, consistent with these observations. To begin elucidating mechanisms regulating the pattern of expression of GDNF, we have cloned the human gene, and characterized the promoter. The promoter is highly GC rich, and lacks canonical CCAT-box and TATA-box motifs. It contains more than 12 binding sites for known transcription factors. These cis-elements have the potential to interact with factors regulating constitutive expression (Sp1) and developmental expression (bHLH). Moreover, the promoter contains sites for binding transcription factors which respond to environmental signals, including CREB, AP2, Zif/268, NFkB, and MRE-BP. Combinatorial actions of these transcription factors may account for the extraordinarily complex expression patterns of the GDNF gene. Importantly, we demonstrate that the hGDNF gene utilizes a promoter distinct from that identified in the rodent GDNF gene, a finding with ramifications for Parkinson's disease and ALS research.

Does considering severity of illness improve interpretation of patient satisfaction data?

With the emergence of new, relatively low-cost code-based severity indexes, this question arises: Do complex descriptions of patient population in terms of severity yield a clearer picture of patients' opinions about hospital care and service? Consumers and third-party payers of healthcare are using patient satisfaction data with increasing frequency to evaluate the quality of care that hospitals provide. Insurers also use satisfaction data, when they are available, for contracting and ensuring provider accountability. The study described here examines whether the all patient refined-diagnosis related groups (APR-DRG) severity-of-illness rating system, in particular, can explain the variability in inpatient satisfaction ratings independently of patient demographics and clinical events. Multiple logistic regression was used on a data set of 3,720 patient records from one tertiary care facility, and model terms were fitted on the basis of reason for admission, year, gender, length of stay, age, and severity. The findings were that age and reason for admission were consistent predictors of high satisfaction on 14 survey items. APR-DRG severity was not a significant factor. Length of stay made a small but significant contribution on three items related to clinical quality.

The rapid L- and D-aspartate uptake in cultured astrocytes.

Astrocytes in primary culture possess a rapid L-aspartate saturable transport system (K(m) = 93 microM; V(max) = 81 nmol/min/mg protein), which shows certain stereospecificity since V(max) was 36% lower for D-aspartate uptake. These are values obtained at short incubation time (15 seconds), to obtain approximate initial rate conditions. Metabolic energy inhibitors, rotenone and iodoacetate very potently inhibited the L- and D-aspartate uptake processes, indicating that the transport process is an active one. However, the accumulation of L-aspartate was "enhanced" by inhibitors of L-aspartate metabolism, such as the aspartate aminotransferase inhibitor, aminooxyacetate and L-methionine sulfoximine, an inhibitor of glutamine synthetase, whereas D-aspartate (a non-metabolizable analog of L-aspartate) uptake was not affected. The accumulated levels of L-aspartate in the presence of aminooxyacetate were similar to the levels of D-aspartate. These effects of L-aspartate metabolic inhibitors, suggest that due to metabolism of the the L-aspartate, short incubation time (eg., 15 seconds) is necessary to measure the initial rate of L-aspartate uptake, in order to obtain the "true" kinetic parameters.

Phospholipase A2 action on planar lipid bilayers generates a small, transitory current that is voltage independent.

Addition of either bee venom or Trimeresurus flavoviridis phospholipase A2 (PLA2) to the solution bathing the front side of a voltage-clamped, planar lipid bilayer consistently produced a transitory current lasting approximately 100 s. This current is consistent with anions moving through the membrane to the rear side. The peak current is independent of holding potential. PLA2 activity on phospholipid membranes not only produced a current but also led to membrane rupture within 300 s. The current depends on Ca2+ and lipid type. Addition of PLA2 in the absence of Ca2+ or to membranes made of nonsubstrate lipids (e.g., glycerol monooleate or lysophosphatidylcholine) produced no current and did not break the bilayer. Peak current height, signal decay time, and time to membrane rupture all depended on PLA2 dose, whereas total charge produced was constant. This current does not flow through ion channels because there are no channels present and the current is not voltage dependent. The evidence is consistent with the hypothesis that the current is generated by the movement of ionized fatty acid produced by PLA2 action. These results demonstrate a simple method to measure enzyme activity in the presence of different substrates and varied environmental conditions.