LP and MC

Buenos Aires, Argentina

LP and MC

Buenos Aires, Argentina
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Vgat-ires-Cre31, Sst-Cre32 and PV-Cre33 knock-in mice (The Jackson Laboratory, Bar Harbour) and C57BL/6 male mice, 10–25 weeks old, were used. Mice were housed under standard conditions in the animal facility and kept on a 12 h light/dark cycle. All procedures were performed in accordance with national and international guidelines, and were approved by the local health authority (Landesamt für Gesundheit und Soziales, Berlin) and the Stanford University Institutional Animal Care and Use Committee. Injections were performed as described previously22, 34. Mice were anaesthetized with isoflurane and placed in a stereotaxic head frame (David Kopf Instruments). A 34-gauge bevelled metal needle connected via a tube with a microsyringe pump (PHD Ultra, Harvard Apparatus) was used to infuse viruses at a rate of 100 nl min−1. After infusion, the needle was kept at the injection site for 10 min and then slowly withdrawn before the incision was sutured. Optogenetic constructs from K. Deisseroth, purchased from Penn Vector Core, UNC Gene Therapy Center Vector Core, or provided by K. Deisseroth, were used. For manipulations of the LS–LH pathway, Vgat-Cre mice were injected bilaterally in the LS (right: anterior-posterior (AP) 0.74, lateral (L) 0.38, ventral (V) 3.3; 3.1 mm; left: AP 0.62, L 0.33, V 3.45; 3.0 mm) with 0.3 μl per injection site of AAV2/5-Ef1a-DIO-ChETA(E123T/H13R)-eYFP-WPRE-hGH (Penn Vector Core, titre 1.75 × 1012 viral genomes (vg) per ml). Sst-Cre mice were injected bilaterally in the LS (right: AP 0.38, L 0.4, DV 3.0, 2.7 mm; left: AP 0.26, L 0.4, V 3.0, 2.5 mm) with 0.125–0.25 μl per injection site of AAV2/5-Ef1a-DIO-ChETA(E123T/H13R)-eYFP-WPRE-hGH, titre 1.75 × 1012 vg ml−1 (Penn Vector Core) or 0.2 μl per injection site of AAVdj-nEF-DIO-NpHR-TS-p2A-hChR2(H134R)-eYFP (eNPAC2.0, titre 6.1 × 1012 vg ml−1) or 0.125–0.2 μl per injection site of AAV2-EF1a-DIO-eYFP-WPRE-hGH (Penn Vector Core, titre 2 × 1012 vg ml−1). For manipulation of LH cells, Vgat-Cre mice were injected bilaterally in the LH (AP −1.5, L ±1, V 5.4 mm) with 0.3 μl per injection site of AAV2/5-Ef1a-DIO-ChETA(E123T/H13R)-eYFP-WPRE-hGH (Penn Vector Core) or 0.3 μl per injection site of AAVdj-nEF-DIO-NpHR-TS-p2A-hChR2(H134R)-eYFP (eNPAC2.0, titre 6.1 × 1012 vg ml−1) or 0.3 μl per injection site of AAV2-EF1a-DIO-eYFP-WPRE-hGH (Penn Vector Core, titre 2 × 1012 vg ml−1). For manipulations of the mPFC–LH pathway, mice were bilaterally injected in the mPFC (AP 1.70, L ±0.35, V 2.85 mm) with 0.25–0.5 μl per injection site of AAV2-CaMKIIa-hChR2(H134R)-eYFP (Penn Vector Core, titre 2.55 × 1012 vg ml−1) or 0.25–0.5 μl per injection site of AAV5-CaMKIIa-ChETA(E123T/H134R)-eYFP-WPRE-hGH (Penn Vector Core, titre 1.26 × 1013 vg ml−1), or 0.25–0.5 μl per injection site (AAVdj-hSyn-NpHR-TS-p2A-hChR2(H134R)-eYFP (eNPAC2.0, titre 2.9 × 1013 vg ml−1) or 0.5 μl per injection site of AAV2-CaMKIIa-eYFP (University of North Carolina Vector Core, titre 5 × 1012 vg ml−1). For CLARITY experiments, mice were injected in the mPFC (AP 2.0, L 0.3, V 2.6 mm) with 1 μl AAV8-CaMKIIa-eYFP-NRN. For synaptophysin imaging, 1 μl AAV8-CaMKIIa-synaptophysin-mCherry (7 × 1013), was injected in the mPFC (AP 2.0, L 0.3, V 2.6 mm). Optic fibre implants were fabricated from 100 μm diameter fibre (0.22 numerical aperture (NA), Thorlabs) and zirconia ferrules (Precision Fibre Products). For optogenetic manipulations of mPFC–LS pathway, mice were implanted with optic fibre implants on top of the LS (right, AP 0.1, L 0.25, V 2.25 mm, left, AP 0.5, L 0.3, V 2.7 mm). For optogenetic manipulations in the LH, optical fibres were bilaterally (for LS–LH and LH stimulation or inhibition) or unilaterally (LH stimulation combined with the LH silicon probe recordings) implanted above the LH (AP −1.6, L 1, V 4.8 mm). Arrays of single tungsten wires (40 μm, California Fine Wire Company), stationary implanted linear silicon probes (CM32, NeuroNexus Technologies), or movable probes (B32 or B64, NeuroNexus Technologies) mounted on a microdrive35 were implanted as described previously22, 26. The following coordinates were used for electrode implantations in the LS: AP 0–0.5, L 0.2–0.45, V 2.3–3.4 mm (B32 probes, B64 probes (mPFC–LS co-implantations), CM32, wire arrays); LH: AP −1.6, L 1, V 4.7 mm (B32 probes, B64 probe, wire arrays); mPFC: AP 1.4–1.9, L 0.3, V 3.0 mm (B64 probes (mPFC–LS co-implantations), wire arrays); dorsal hip: AP −2.1, L 1.6, V 1.5 mm (wire arrays), ventral hip: AP −3.16, L 2.5–3.5, V 4.0 mm (wire arrays). Reference and ground electrodes were miniature stainless-steel screws in the skull above the cerebellum. The implants were secured on the skull with dental acrylic. Electrodes were connected to operational amplifiers (HS-8, Neuralynx, or Noted BT) to eliminate cable movement artefacts. Electrophysiological signals were differentially amplified, band-pass filtered (1 Hz–10 kHz, Digital Lynx, Neuralynx) and acquired continuously at 32 kHz. A light-emitting diode was attached to the headset to track the mouse’s position (at 25 Hz). Timestamps of laser pulses were recorded together with electrophysiological signals. A 3-m-long fibreoptic patch cord with protective tubing (Thorlabs) was connected to a chronically implanted optical fibre with a zirconia sleeve (Precision Fibre Products), which allowed the mice to explore an enclosure freely or perform a behavioural task during optogenetic manipulations. Subjects were randomly assigned to the experimental conditions. For optogenetic stimulation, the patch cord was connected to a 473-nm diode-pumped solid-state laser (R471005FX, Laserglow Technologies) with an FC/PC adaptor. The laser output was controlled using a stimulus generator and MC_Stimulus software (Multichannel Systems). Optogenetic stimulation of LS–LH and mPFC–LS projections consisted of 5 ms blue (473 nm) light pulses, at 66.7 Hz or a control, non-gamma (theta) intensity-matched stimulation (167 Hz bursts of 4 ms pulses repeated at 9 Hz) with the light power output (during light-on parts of illumination cycles) of 10–25 mW from the tip of the patch cord measured with a power meter (PM100D, Thorlabs). Optogenetic stimulation of LH somata consisted of 5 ms blue (473 nm) light pulses, at 20 Hz, with light power output (during light-on parts of illumination cycles) of 10–25 mW from the tip of the patch cord. For bilateral optogenetic inhibition, optic fibre implants were connected via patch cords to a 593-nm diode-pumped solid-state laser (R591005FX, Laserglow Technologies) using a multimode fibre optic coupler (FCMM50-50A-FC, Thorlabs), continuous yellow light, approximately 20 mW from the tip of each patch cord. Duration of light delivery is described below for each type of behavioural experiment. For control experiments, mice expressing YFP in the same brain regions (mPFC or LS) were used, and optostimulation was performed as described above. For within-animal comparisons in sessions in which food intake was measured during LH stimulation (Extended Data Figs 2i, 8c, d, f), optic patch cords were connected to dummy ferrules, attached to the headset, and light of the same wavelength and power as during opsin-activating stimulation was delivered. Free-access feeding model. This was performed in a chamber similar to that described previously36 (Fig. 1a). Mice freely explored a custom two-chamber (30 × 50 × 20 cm) enclosure, which contained food and water in designated areas (each area 10 × 10 cm; see Fig. 1a). Food (Dustless Precision Pellets, 20 mg, Rodent Purified Diet, Bio Serv) was provided either in a food cup or in a pellet feeder (Coulbourn Instruments Pellet Feeder H14-23M; sampling rate 10 Hz, one nose poke led to the delivery of one food pellet). Before experiments, mice received these pellets in the homecage for at least 2 days, and were habituated to the behavioural setup for at least 3 days. Coordinates of 10 × 10 cm food, drinking, non-food corner zones and a control zone located in the non-food compartment were defined. Times of entering and leaving each zone were extracted from the mouse’s position-tracking data. An approach rate was defined as the distance between a position of the mouse and the centre of the food zone, the drinking zone or the control zone, divided by the time it took to enter a respective zone. For each experiment, a corner zone, one of which was a food zone, visited first after the onset of stimulation was detected. Latency to enter each zone was defined as the time between the beginning of optogenetic stimulation and the first entry of the mouse into a zone, with the mouse staying in the zone for at least 1 s. To account for differences in distances to a zone after the stimulation onset, in each experiment we have normalized the latency after stimulation onset to the average latency of entering the same zone from the same distance during the baseline. Duration of experimental sessions and optogenetic manipulations are described below. Optogenetic activation of LS –LH projections. Mice explored the enclosure for 30 min: 10 min before stimulation, 10 min during optogenetic stimulation, 10 min after stimulation. Blue light (473 nm) was bilaterally delivered over LS–LH projections, in 5 ms pulses at 66.7 Hz or using a control, non-gamma (theta) intensity-matched stimulation (167 Hz bursts of 4 ms pulses repeated at 9 Hz), with light power output of 10–25 mW. For brief gamma stimulation, 5 ms pulses at 66.7 Hz were delivered for 30 s, followed by a break of 2 min, during a 10 min period. Optogenetic inhibition of LS –LH projections during food approach. Mice explored the enclosure for 30 min. Each time the mouse crossed the border of a food-approach area (20 × 20 cm, marked as an orange dotted line on Extended Data Fig. 6e), continuous yellow (593 nm) light was bilaterally delivered over LS–LH projections. Light delivery stopped each time a mouse left the approach zone. Optogenetic activation of LS –LH projections during free-access to high-fat food. Mice explored the enclosure for 20 min. Blue light (473 nm) was bilaterally delivered over LS–LH projections, in 5 ms pulses at 66.7 Hz, with light power output of 10–25 mW. High-fat food pellets (Testdiet, 60% energy from fat) were weighted before and after the experiment, to calculate the amount of food (>5 mg) consumed per session. Optogenetic stimulation of LH cells. Mice explored the enclosure for 30 or 60 min: 10 or 20 min before stimulation, 10 or 20 min during optogenetic or control light stimulation, 10 or 20 min after stimulation, Dustless precision pellets (BioServ) were counted to measure the amount of food (>1 pellet) consumed per session. Blue light (473 nm) was delivered bilaterally, in 5 ms pulses at 20 Hz. Optogenetic inhibition of LH cells in food-deprived mice. Mice received approximately 2.5–3.0 g of standard chow daily; the mouse weight was controlled and weight loss did not exceed 10%. Dustless precision pellets (BioServ) were counted to measure the amount of food consumed by hungry mice (>3 pellets in baseline) per session. The experiments consisted of four epochs: 10 min light-on (optogenetic or control stimulation), 10 min light-off, 10 min light-on, and 10 min light-off. This was performed in a custom two-chamber enclosure similar to the one used for the free-feeding model (30 × 50 × 20 cm). One of the chambers contained a familiar object, whereas the other contained a new object. Before experiments, mice were habituated to the enclosure containing two objects, then for each experimental session one of the objects (new object) was replaced, whereas the object in the other chamber (familiar object) remained the same. Optogenetic stimulation started as the mouse was put in the enclosure. Mice freely explored the enclosure maximally for 2 min, otherwise a session was finished once the mouse visited both objects. Blue light (473 nm) was bilaterally delivered over LS–LH projections, in 5 ms pulses at 66.7 Hz, with light power output of 10–25 mW. Spatial non-matching to place testing on the T-maze was performed as described elsewhere37. The T-maze (start arm: 46 × 11 × 10 cm, choice arm: 80 × 11 × 10 cm; see Fig. 4p) was made of pieces of wood painted dark-grey. For spatial non-matching to place testing, each trial consisted of a sample run and a choice run. During the sample run, mice could run only to one arm (left or right, according to a pseudorandom sequence with equal numbers of left and right turns per session) because another arm was blocked by a wooden block. A reward (0.1 ml condensed milk or a 20 mg food pellet) was available in the food well at the end of the arm. After the sample run, mice stayed in another, familiar, enclosure for 10–15 s. The block was then removed, and mice were placed at the end of the start arm to perform the test run. Mice were rewarded for choosing the previously unvisited arm (that is, for alternating). For this test and all subsequent experiments, entry into an arm was defined as when a mouse had placed all four paws into the arm. Mice ran one trial at a time with inter-trial intervals of 3–5 min. Each mouse conducted 20–40 trials in total (10 trials per day). For a subset of experiments, mice were water-restricted and water was used as a reward instead of food. A number of slow (30–60 Hz) or fast (60–90 Hz) gamma-oscillation episodes (detected as described below) in the start or choice arm was normalized by dividing by the mean number of gamma events in each arm during the whole experiment. Optogenetic activation or inhibition of mPFC–LS projections in the T-maze. Optogenetic stimulation started as the mouse was put at the end of the start arm, and finished when the mouse reached the reward. Blue light was delivered on mPFC–LS projections in 5-ms pulses at 66.7 Hz, or in a non-gamma (theta) intensity-matched stimulation protocol, described above for free-feeding model. For inhibition, yellow light was delivered onto mPFC–LS projections continuously during the run. LFP was obtained by down-sampling of the wide-band signal to 1,250 Hz using Neurophysiological Data Manager38 (http://neurosuite.sourceforge.net/). Gamma oscillations were detected at 30–60 Hz, 60–90 Hz and, for the analysis shown in Extended Data Fig. 1i, 90–120 Hz, bandpass filtered, rectified and smoothed with a 15-ms window LFP signals. Events with amplitudes exceeding 2 s.d. above mean for at least 25 ms were detected13. The beginning and the end of oscillatory epochs were marked at points at which the amplitude fell below 1 s.d. Power spectral density and coherence were computed using the multitaper method (NW = 3). For the analysis of association between gamma power and approach rate, the cumulative power in the 30–60 and 60–90 Hz bands as well as the approach rate (see ‘Behavioural assays’) was computed, and for each 1-s recording epoch, gamma power was z-transformed. Values within 10 s before entry in the food or drinking zones were statistically evaluated. Current source density (CSD) maps (versus time and depth) were computed as previously described37, 39. LFP depth profiles, recorded using CM32 probes with the spatial sampling of 100 μm, were averaged using peak gamma oscillations detected in an LS channel as triggers. The second spatial derivative of the obtained voltage traces, that is, CSD, indicates locations of current sinks and sources in the extracellular space40. For the analysis of mPFC–LS and hippocampus–LS coherence, normalized current flow density in the LS was computed by subtraction of gamma-band filtered LFP signals, recorded by a pair of wire electrodes in the LS against a common screw-reference above cerebellum40, 41. Action potentials were detected in a high-pass filtered signal using NDManager16 (http://neurosuite.sourceforge.net/). Spike waveforms were extracted and represented by the first three principle components and by amplitudes of action potentials. Spike sorting was performed automatically using KlustaKwik42 (http://klusta-team.github.io/klustakwik/) followed by manual clusters adjustment using Klusters38. Isolation distance42 was computed for sorted units (LH: 101.5 ± 8.0, LS: 66.3 ± 4.6, mPFC: 56.3 ± 3.6). Phase of gamma oscillations was computed for signal epochs within detected gamma episodes as described elsewhere37, 43. In brief, 0° and 360° were assigned to troughs of each gamma cycle and 180° to a cycle peak, phases for each data sample between these points were computed using linear interpolation13, 37. Subsequently, gamma phases were obtained for data samples when action potentials were emitted, for each recorded neuron, and firing phase histograms were computed. A possible asymmetry of oscillation cycles leads to a different number of phase samples composing ascending and descending parts of the cycle and can bias firing phase histograms39. To prevent this, we tested uniformity of grand gamma phase distributions for each recording using the Rayleigh test and, if significantly non-uniform, computed a deviation of a grand phase histogram from uniformity, via division by the average across all bins. In such recordings, firing histograms were normalized by the corrected grand phase histogram37, 44, 45. Each firing phase histogram was normalized by its total number of spikes. Circular uniformity, mean phase and the resultant vector length were estimated for each histogram. Before averaging, individual histograms were convolved with the Gaussian kernel46 of size 0.65 s.d. Putative LH neurons were optogenetically identified based on rapid (<10 ms lags in laser pulse onset-triggered cross-correlations, computed with 1-ms bins) increase of firing after onset of laser pulses. Reliability of light-induced responses was estimated as a probability of the maximal light-induced spike count in a Poisson distribution computed for cross-correlogram (CCG) delays in the pre-pulse baseline26, 47. To estimate gamma-rhythmic responses of LH cells to LS–LH stimulation, a cross-correlation (CCG) with the times of LS–LH light stimulation was computed for each cell. To avoid spurious CCG peaks at the stimulation frequency, every second time stamp of light was used as a trigger. A reshuffled CCG was computed using light times shifted to a baseline, light-off, recording epoch. A power spectrum of the response of a cell was then obtained by subtracting the power spectrum of the reshuffled CCG from the power spectrum of the stimulation CCG. Firing of LH neurons in the free-access feeding model was evaluated as described elsewhere for quantification of positional firing37, 48. Firing maps were computed by dividing the number of spikes in a given spatial pixel (2 × 2 cm) by the time spent in this pixel. Periods of immobility (speed <3 cm s−1) were excluded from the analysis. Peak firing rate was defined as the maximum firing rate over all pixels in the environment. For calculation of food-zone preference (FZ-match index), the average firing rate of a cell in the food zone was divided by the average firing rate in a control zone of the same size (10 × 10 cm), located in the non-food compartment of the enclosure. For the analysis of firing during gamma oscillations, cells were split in ‘FZ-match’ or ‘FZ-mismatch’ groups based on an FZ-match index higher or lower than 1, respectively. For identification of LH cells, excited in response to LH or LS –LH optogenetic stimulation, the number of spikes (x) was computed for each 100-ms bin during the baseline (10 min) and within 3 s after stimulation onset. A distribution derived from the baseline was fitted to a Poisson distribution, and rate parameter λ was estimated. A bin with maximal count of spikes during stimulation was assigned to observed value x . P value was defined as P(x ≥ x ), in which x follows Pois(λ). The firing rate ratio (R) between 3 s baseline and stimulation epochs was computed for each stimulation epoch and then averaged across stimulation epochs. A cell was classified as excited if P < 0.05 and R > 1. Code is available from the corresponding authors upon request. Each statistical test was used according to the design of the experiment and the structure of the data. Two-group comparisons were performed using t-test, Mann–Whitney or Wilcoxon matched-pairs tests depending on the normality of a distribution. Assessment of effects in experiments involving several conditions was performed using ANOVA, followed, when appropriate, by Bonferroni (for pre-selected contrasts) or Tukey tests, adjusting for multiple comparisons. Grubbs’ test was used to exclude outlier points from behavioural datasets. Depending on the normality of a distribution, Pearson’s or Spearman’s correlations were computed. For group comparisons, two-tailed statistical tests were applied. Sample size was determined according to the accepted practice for the applied assays, no statistical methods were used to predetermine sample size. Conditions of the experiments were accounted during design of analysis algorithms, computations were subsequently performed blindly using automatic selection of data from a database. A detailed description of statistical analysis is provided in the statistical section of the Supplementary Information. Descriptive statistics are reported as mean ± s.e.m. Primary cultured neurons were prepared from the hippocampi of P Sprague–Dawley rat pups (Charles River Laboratories), as described previously49. CA1 and CA3 hippocampal regions were taken out and digested with 0.4 mg ml−1 papain (Worthington), and plated onto 12-mm glass coverslips that were pre-coated with 1:30 Matrigel (Beckton Dickinson Labware). Cultures were kept under neurobasal-A medium (Invitrogen) containing 1.25% FBS (HyClone), 4% B-27 supplement (Gibco), 2 mM glutamax (Gibco) and 2 mg ml−1 fluorodeoxyuridine (FUDR, Sigma) and plated at a density of 65,000 cells per well in 24-well plates. The plates were incubated at 37 °C in a humid incubator with a constant level of 5% CO . Cultured neurons were transfected at 6–10 days in vitro (DIV). A DNA–CaCl mix composed of the following was prepared for transfection per well: 1 μg of endotoxin-free DNA for recordings, 1.875 μl 2 M CaCl , and sterile H O for a total volume of 15 μl. Another 15 μl of twice-filtered HEPES-buffered saline (HBS, in mM: 50 HEPES, 1.5 Na HPO , 280 NaCl, pH 7.05 with NaOH) for each DNA–CaCl mix. This mix was incubated at room temperature for 20 min. In the meantime, the neuronal growth medium was removed from the wells and kept at 37 °C, and replaced with 400 μl pre-warmed minimal essential medium (MEM). After incubation of the DNA–CaCl –HBS mix was complete, the mix was then added dropwise into each well, and plates were incubated for 45–60 min at 37 °C. Once the transfection was complete, each well was washed three times with 1 ml of pre-warmed MEM. The MEM was then replaced with the original neuronal growth medium, and plates were placed into the culture incubator at 37 °C. Whole-cell patch-clamp recordings of cultured hippocampal neurons were performed 3–5 days after transfection with the construct AAVdj-hSyn-NpHR-TS-p2A-hChR2(H134R)-eYFP (eNPAC2.0). Expression of the construct was identified by eYFP fluorescence. The external recording medium was composed of the following (in mM): 125 NaCl, 2 KCl, 25 HEPES, 2 CaCl , 2 MgCl , 30 d-(+)-glucose. pH 7.3, with synaptic transmission blockers d-2-amino-5-phosphonovaleric acid (AP5; 25 μM), 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX; 10 μM), and gabazine (10 μM). The intracellular recording solution contained (in mM): 130 potassium gluconate, 10 KCl, 10 HEPES, 10 EGTA and 2 MgCl . An upright microscope (BX61WI, Olympus) with infrared differential interference contrast (IR-DIC) was used for visualization and recording of the expressing neurons. A Spectra X Light engine (Lumencor) attached to the fluorescent port of the microscope was used for light application, for detecting eYFP expression and for blue or yellow light delivery for opsin activation. A 475/28 nm and a 586/20 nm filter (Chroma) were used for blue and yellow light respectively (Chroma). A power meter (ThorLabs) was used to measure the light power through the microscope objective, and light power density was set at 5 mW mm−1 (ref. 49). Recordings were obtained using a MultiClamp700B amplifier, 1440A Digidata digitizer, and pClamp10.3 software (Molecular Devices). Data were analysed with pClamp10.3 and SigmaPlot (SPSS). Photocurrent amplitudes at blue and yellow were measured at steady-state at the end of a 1-s light stimulation protocol. To measure spike inhibition probability at 586 nm, we first applied a 50–200 pA electrical current injection (depending on spike threshold of the recorded cell) to induce spiking in the expressing neurons. Spike inhibition probability was calculated as the percentage in which yellow light application inhibited spiking during the electrical current injection. To measure spike generation probability with blue light, we applied 5-ms width pulses of 475 nm light at 5 or 20 Hz frequency, and calculated the percentage of action potentials generated by the blue light pulse train. Series resistance was carefully monitored for stability throughout the recordings. To ensure accurate measurements of voltage-clamp recordings, data were incorporated for analysis only if the series resistance was below 25 MΩ and changed less than 20% throughout the recording. Standard whole-cell slice patch-clamp recordings were undertaken after slice preparation of at least 2-month-old mice. In brief, after gluing a block of brain with cyanoacrylate glue to the stage of a Campden Vibroslice, coronal brain slices of 250-μm thickness containing the LH were cut while immersed in ice-cold slicing solution. Slices were incubated for 1 h in artificial cerebrospinal fluid (ACSF) at 35 °C then transferred to a submerged-type recording chamber. Living neurons containing fluorescent markers were visualized in acute brain slices with an upright Olympus BX61WI microscope equipped with an oblique condenser and appropriate fluorescence filters. After identifying appropriate neurons by their fluorescence, oscillatory currents of 10 pA amplitude (30, 50, 70 and 100 Hz) were injected for 5 s to the cell during whole-cell patch-clamp recordings. Recordings of membrane potentials were analysed in MatLab. To record selectively from Vgat neurons, a cross between Vgat-ires-Cre and CAG-tdTomato mice33 was used. To target MCH-Cre neurons selectively, MCH-Cre mice were injected into the LH (1.3 mm caudal from bregma; ±0.95 mm lateral from midline; and 5.25 and 5.15 mm ventral from brain surface) with a Cre-dependent ChR-mCherry. For brain slice recordings, ACSF and ice-cold slicing solution were gassed with 95% O and 5% CO , and contained the following (in mM) ACSF: 125 NaCl, 2.5 KCl, 1 MgCl , 2 CaCl , 1.2 NaH PO , 21 NaHCO , 2 d-(+)-glucose, 0.1 Na+-pyruvate and 0.4 ascorbic acid. Slicing solution: 2.5 KCl, 1.3 NaH PO.H 0, 26.0 NaHCO , 213.3 sucrose, 10.0 d-(+)-glucose, 2.0 MgCl and 2.0 CaCl . For standard whole-cell recordings, pipettes were filled with intracellular solution containing the following (in mM): 120 K-gluconate, 10 KCl, 10 HEPES, 0.1 EGTA, 4 K ATP, 2 Na ATP, 0.3 Na GTP and 2 MgCl , pH 7.3 with KOH. Multitaper power spectra of voltage traces and of injected current traces were computed and divided, resulting in impedance spectra. Mean impedance at ±1.5 Hz around stimulation frequency was computed. Brain hemispheres were clarified using the CLARITY procedure as described elsewhere27. In brief, a brain hemisphere was fixed in hydrogel solution (4% PFA, 1% acrylamide/bis) for 72 h at 4 °C. After polymerization (37 °C, 4 h), the brain hemispheres were clarified in 8% SDS for 8 days (at 40 °C), then washed three times with PBST (0.2% Triton X-100) for a total of 24 h at 37 °C. Hemisphere images were acquired with the Ultramicroscope II (Lavision Biotec)27. Samples were mounted to a custom 3D printed holder using RapidClear Mounting Gel (Sunjin laboratory). Brains were imaged using a 2×/0.5 NA objective at 0.8× zoom using a single light sheet illuminating from the dorsal side of the sample. Z-step was set to 4 μm. Fourteen horizontal focal points were set to each imaging plane to create a homogeneous field of view. For synapsin staining the brains were cut, after CLARITY processing, into 1-mm-thick sections. Primary antibody: rabbit anti-synapsin (Cell Signaling, 5297), 1:400, in 0.3% PBST, room temperature, 24 h; secondary antibody: donkey anti-rabbit (Alexa 594, Jacksonimmuno), 1:200, in 0.3% PBST, room temperature, 24 h; then sections were refractive index-matched and mounted in RapidClear CLARITY-specific gel (Sunjin Laboratory). Sections were imaged at bregma = 0.5 using Olympus FV1200 confocal, 40×, 1.3 NA, oil objective, at 4× zoom. For synaptophysin imaging, brains expressing CaMKIIa-synaptophysin-mCherry in the mPFC were cut into 0.5-mm-thick section for CLARITY clearing and imaging. The sections were imaged at bregma = 0.5 using Olympus FV1200 confocal, 40×, 1.3 NA, oil objective, at 4× zoom. After completion of the experiments, mice were deeply anaesthetized and electrolytic lesions at selected recording sites were performed. Subsequently, the mice were perfused intracardially with saline followed by 4% paraformaldehyde in PBS and decapitated. Brains were fixed overnight in 4% paraformaldehyde, equilibrated in 1% PBS for an additional night and finally cut into 40 or 50 μm slices using an oscillating tissue slicer (EMS 4500, Electron Microscopy Science). Brain slices were mounted (Flouromount Aqueous Mounting Medium, Sigma-Aldrich). Images were taken using an Olympus BX 61 microscope (×2/0.06 NA, ×10/0.3 NA and ×20/0.5 NA, dry) or using a Leica DMI 6000 microscope (×20/0.7 NA, ×63/1.4 NA; oil-immersion objectives). All data generated or analysed during this study are either included in this published article or are available from the corresponding authors on reasonable request.


News Article | November 22, 2016
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Two healthcare facilities developed by NexCore Group, a national healthcare real estate developer, have been honored as Finalists in Healthcare Real Estate Insights™ magazine’s annual, national awards program. NexCore, which is headquartered in Denver, developed the facilities in Avon and Lewis Center. The HREI Insights Awards™ are the only national awards dedicated to recognizing excellence in the areas of healthcare real estate (HRE) development and executive leadership. The awards are presented by Healthcare Real Estate Insights™ magazine, the first and only national magazine entirely devoted to covering HRE development, financing and investment. Todd Varney, NexCore Managing Director Development and Acquisitions, noted, “We are very pleased that HREI magazine has honored two of NexCore’s projects – Buck Creek Medical Plaza and MC Fitness & Health – as Finalists in their prestigious annual awards program. NexCore always strives to help our clients realize their goals with high quality, cost-effective and efficient facilities that deliver excellent healthcare services in the communities they serve. These two facilities embody the realization of those goals.” NexCore projects were named Finalists seven times and a Winner one time in the three previous years of the highly competitive HREI Insights Awards™ program, whose results are announced each December. The NexCore-developed projects that were named 2016 Finalists are: A Finalist in the “Best New Medical Office Buildings (MOBs) and Other Outpatient Facilities (25,000 to 49,999 square feet)” Category, Buck Creek Medical Plaza is a $28 million, three-story, 48,300 square foot MOB and freestanding emergency department (FED)/urgent care center located in the heart of the Vail Valley. The facility, which opened in June 2016, realized the longstanding, elusive goal of a new, one-stop outpatient center in the Valley, further complicated by unique geography, topography and extremely high concentration of government-owned land. In a unique collaboration, NexCore was able to secure the perfect 2.15-acre parcel through an ingenious and exemplary land swap that involved the Eagle River Fire Protection District, the City of Avon, and New York financier and Vail resident Oscar Tang, an individual highly committed to the Vail Valley, who owned adjacent land. NexCore fast-tracked its process and broke ground on Buck Creek seven months later. With anchor tenants Centura Health and Colorado Mountain Medical, and other specialties such as physical therapy, dental and allergy, top-quality healthcare is being delivered to a Colorado mountain community that has been especially challenged by the changes in medical care during the past few years. Buck Creek Medical Plaza’s centralized location with great visibility, beautiful views, access from U.S. Interstate 70 and free parking provides the kind of care the Vail Valley community demanded and deserved, while further establishing the area as a top-notch resort and residential community with quality healthcare, physicians and diversified jobs. With its vibrant mix of providers, Buck Creek Medical Plaza was 94 percent leased at opening. The service mix creates a built-in referral network for tenants and is projected to generate 88,000 annual patient visits. A Finalist in the “Best New MOBs and Other Outpatient Facilities (100,000 square feet or more)” Category, MC Fitness & Health is a $40 million, three-story, 130,000 square foot MOB, hospital outpatient clinic, medical fitness center and FED in the fast-growing Columbus, Ohio, suburb of Lewis Center. Mount Carmel Health System (MCHS) wanted to develop an outpatient center to protect and increase its market share in the economically thriving area in Delaware County. The new facility would be part of the MCHS “hub and spoke” ambulatory network, feeding Mount Carmel St. Ann’s Hospital in Westerville about eight miles away. MCHS also envisioned the facility as a community center focused on wellness that would signal the system’s move toward value-based care. MCHS identified Lewis Center and the U.S. 23 corridor as the ideal area for the facility, with its great visibility/branding, ease of patient access, future growth opportunities and a strategic location in a growing suburban retail corridor. NexCore led the site acquisition process, including market demand analysis, site identification, site due diligence, site selection and land closing. NexCore helped MCHS develop its program for the outpatient/medical fitness center, refine space requirements, and right size the facility. Six months into construction, MCHS wanted to change the urgent care center to an ED to accommodate service needs in Delaware Co. NexCore’s team redesigned the interior space, reengineered mechanical/electrical systems, priced construction and presented a proposal to MCHS in 37 days. MCHS accepted the proposal, and NexCore fast-tracked development of the ED, which opened just 45 days after the rest of the facility. NexCore facilitated early collaboration on program and design for the facility, resulting in efficient use of space and funds. They were able to reduce the required land area from 13 to 11 acres, thereby reducing land and site development costs by over $600,000, which translated to lower lease rates for MCHS and other tenants. NexCore helped MCHS bring a one-of-a-kind outpatient facility to Lewis Center/Delaware County, one that offers patients a complete continuum of care (prevention, diagnosis, treatment, retail services) and responds to healthcare reform. Since the facility opened, MCHS has implemented many community engagement and population health management initiatives, including a monthly lecture series, hosted events (cook-out and pool party, several 5K races, and Women’s Day), partnerships with local schools and recreational teams (swim meets, mentor program and volunteer program for developmentally disabled students), participation in SAFE Coalition for Delaware County and Partnership for a Healthy Delaware County, and corporate/senior discounts. The HREI Insights Awards™ will be presented live Dec. 8 at the RealShare Healthcare Real Estate Conference in Scottsdale, Ariz. For more information on the awards, please see http://www.HREInsights.com. NexCore Group develops healthcare facilities for hospitals, health systems and physicians. We solve complex real estate challenges through innovative building solutions and creative financial structures. In a rapidly changing healthcare industry, NexCore has the superior expertise to deliver quality environments and flexible spaces that help grow your business, lower expenses and mitigate risk. Over the past 25 years, NexCore has developed and acquired a total of 6.1 million square feet of health care facilities across the country. Our company has been recognized repeatedly as one of the Top Healthcare Real Estate Developers in the U.S. by Modern Healthcare magazine. NexCore is headquartered in Denver, Colorado, and as the company continues to grow so does its geographic reach. Over the last several years, NexCore has opened regional offices in Chicago; Bethesda, Md.; Portland, Ore.; Dallas; Orlando, Fla.; and Laguna Niguel, Calif. For more information, please visit http://www.nexcoregroup.com. Contact: Tracy Hindman, Marketing Director, 303-293-0673.


NEW YORK, Nov. 23, 2016 (GLOBE NEWSWIRE) -- Motif Bio plc (Motif) (NASDAQ:MTFB), (NASDAQ:MTFBW), a clinical stage biopharmaceutical company specializing in developing novel antibiotics, announced today the closing of its initial U.S. public offering of approximately 2.44 million American Depositary Shares (ADSs) with 50% warrant coverage. Each ADS, representing 20 ordinary shares of Motif, and warrant to purchase 0.5 ADSs were sold at a price to the public of $6.98 per ADS and warrant combination.  Motif has granted the underwriters a 30-day option to purchase up to an additional 292,618 ADSs and/or 146,309 warrants to cover over-allotments, if any, in the U.S. offering. Each full ADS warrant has a per ADS exercise price of $8.03. The ADS warrants are exercisable immediately and have a term of five years. The ADSs and ADS warrants are listed on The NASDAQ Capital Market under the symbols “MTFB” and “MTFBW,” respectively. H.C. Wainwright & Co., LLC acted as the sole book-running manager for the offering. Motif also closed its concurrent placement in Europe of approximately 22.9 million ordinary shares with 50% warrant coverage. Each ordinary share and warrant to purchase 0.5 of an ordinary share were sold at a price to the public of 28 pence per ordinary share and warrant combination. Each full ordinary share warrant has a per ordinary share exercise price of 32.2 pence.  The ordinary share warrants are exercisable immediately and have a term of five years.  Motif’s ordinary shares trade on the AIM market of the London Stock Exchange under the ticker symbol “MTFB.” Zeus Capital Limited, Northland Capital Partners and MC Services acted as the placing agents in the European Placement. The aggregate gross proceeds to the Company, before deducting underwriting discounts and commissions, placing agent commissions and other estimated offering expenses, were approximately $25 million. The Company intends to use the net proceeds from these offerings, together with cash and cash equivalents on hand, (i) to fund the expenses to be incurred in conducting the two Phase 3 clinical trials of iclaprim for the treatment of ABSSSI, including the completion of our REVIVE-1 trial; and (ii) for working capital, general and administrative expenses, research and development expenses, and other general corporate purposes. While the Board believes that, along with the Company’s existing cash and cash equivalents, the net proceeds from the U.S. offering and concurrent European placement will provide sufficient capital to enable the Company to complete the REVIVE-1 trial, the Company will require additional funds to complete the REVIVE-2 trial and plans to raise the additional capital through public or private financings and/or other partnering opportunities. The registration statement relating to these securities was declared effective by the U.S. Securities and Exchange Commission on November 17, 2016. The U.S. offering is being made only by prospectus. Copies of the final prospectus related to the offering may be obtained from: H.C. Wainwright & Co., 430 Park Avenue, New York, NY 10022, telephone: 212-356-0500, or e-mail: placements@hcwco.com. Investors may also obtain these documents at no cost by visiting the SEC's website at http://www.sec.gov. This press release shall not constitute an offer to sell or the solicitation of an offer to buy, nor shall there be any sale of these securities in any state or jurisdiction in which such offer, solicitation or sale would be unlawful prior to registration or qualification under the securities laws of any such state or jurisdiction. Motif Bio is a clinical-stage biopharmaceutical company, engaged in the research and development of novel antibiotics designed to be effective against serious and life-threatening infections in hospitalized patients caused by multi-drug resistant bacteria. Our lead product candidate, iclaprim, is being developed for the treatment of acute bacterial skin and skin structure infections (ABSSSI) and hospital acquired bacterial pneumonia (HABP), including ventilator associated bacterial pneumonia (VABP), which is often caused by MRSA (methicillin resistant Staphylococcus aureus). We are currently enrolling and dosing patients in two global Phase 3 clinical trials (REVIVE-1 and REVIVE-2) with an intravenous formulation of iclaprim, for the treatment of ABSSSI.  Data readout for REVIVE-1 is expected in the second quarter of 2017 and REVIVE-2 is on track for data readout in the second half of 2017.


News Article | March 24, 2016
Site: phys.org

Oakland is preparing for a different onslaught - an influx of tech workers and tech companies. One might make the case that Oakland would be very lucky indeed if such a thing happened. After all, cities around the world lovingly tend to anything tech sprouting up in strip malls and suburban garages. Instead, the city - known more for Occupy, the Warriors and MC Hammer than coding - is looking to what has happened to San Francisco in terms of population, culture and cost (cue the head shake and sigh, "It's too late for San Francisco") and asking how Oakland can be different. Can it welcome newcomers without losing its soul? Oakland is at "an inflection point where we have an opportunity to write a new narrative of what the tech industry can be and put out a counternarrative to Silicon Valley and tech," said Catherine Bracy, the managing director of a new organization called the TechEquity Collaborative, which now counts about 15 firms as members. "There's another way to do the business of the Internet." One of the best parts - though some call it one of the worst - of the Bay Area tech boom is that it is decentralized. Opportunity and prosperity are spread out geographically. About 20,500 tech workers live in the East Bay city, home to Pandora and Ask.com, according to the Oakland Metropolitan Chamber of Commerce and Beacon Economics. City leaders estimate that there are about 6,500 tech jobs within the city. About 7.6 percent of Oakland's tech workers are African-American, much higher than the industry standard. Tech businesses in the city have received $830 million in venture funding since January 2014, putting Oakland at 11th of 22 Bay Area cities, according to PitchBook, a private financial market data provider. Still, it's been easy to feel that the tech boom was largely happening elsewhere. Consider that $5.2 billion went to San Francisco companies and Palo Alto firms nabbed $1.2 billion. But when Uber announced it had bought an old Sears building and would house 2,000 to 3,000 employees there, a shock went through the city. Residents began talking about the pros and cons of playing a bigger part in the Bay Area tech ecosystem. Rising housing costs, of course, is the big worry. And job creation is great, but who gets hired? "We look at San Francisco as a cautionary tale," Oakland Mayor Libby Schaff said. The kind of tech hub Oakland wants to be is one with a social conscience, she added. The mayor has touted the idea of "techquity." "I'm determined to do everything in my power to demonstrate that we can take this boom in Oakland and use it to lift up rather than push out our longtime vulnerable residents and our unique culture," she said. At the same time, "cities are dynamic places, they are destined to change. I do not believe in building a wall around my city and preventing people from coming here." Bracy, who has worked at Code for America and the 2012 Obama presidential campaign in California, said the idea of the collaborative is that member companies would commit to a set of standards around the concept of "techquity." While the details are still being worked out, some of those standards might include a commitment to hiring a diverse workforce, identifying future jobs that locals might be trained to do, using local services and playing a larger role in the community. "Equity is a filler word to mean a lot of things," said Darrell Jones, head of business development at Clef, an Internet security firm based in Oakland. Every Wednesday, Clef holds a community dinner at The Port Workspaces where workers from local tech startups come and meet. Tech needs to be "mindful to how they negatively impact this community," he said. Most companies "are externalizing that cost without thinking about it." Jones was asked how his firm of seven, right in downtown Oakland, might be having a negative impact on the area. Engineers with headphones on worked hard at their desks underneath a sign that read, "Treat others the way that they would like to be treated." The harm, he argues, happens in the aggregate, when a lot of companies like his move in and then take over. Could the techquity conversation scare off potential businesses? "We have an opportunity to move the needle for full and inclusive participation in tech," said Barbara Leslie, chief executive and president of the Oakland chamber. "We can learn from our neighbor's tech explosion and position Oakland for growth, while at the same time preserving our identity. The techquity conversation is important to have now." That brings us to Uber. One of the most interesting questions will be how the ride-booking firm integrates with Oakland when it opens its offices in 2017. The company, with help from the mayor's office, has been on a "listening tour" of sorts, meeting with community members and trying to figure out how it wants to be Uber in Oakland. Connect with the local art, music and food scenes? Reflect the city's eclectic range of neighborhoods and its history? "Oakland is going through revitalization on its own," said Laura Zapata, an Uber spokeswoman. "We want to be thoughtful about how we partner with local leaders and want to add value as responsible neighbors." I am torn between appreciating Oakland leaders for their push for "tech equity" and feeling skeptical that getting these commitments will accomplish much. Also, I don't want potential businesses to be scared off by the techquity rhetoric. Oakland might not be the right city for every tech firm but it has the potential to evolve as a tech hub in a more inclusive way. Those who decide to make it home know they are in for more than nice offices, with good proximity to transportation and restaurants. Will they be Oaklandish? Explore further: NY mayor launches bid to help tech startups


The length of time donor lungs could be preserved prior to transplant could be safely extended to more than 12 hours--more than double the average 5-6 hour standard time [1]--without jeopardising recipient outcomes, by using a combination of cold preservation and a new technique called ex-vivo lung perfusion (EVLP), whereby the lung is kept alive outside the body and supported by a supply of oxygen and nutrients. The new study, published in The Lancet Respiratory Medicine, found that patients who received a donor lung preserved for more than 12 hours had similar survival at 1 year post transplant to those who received lungs preserved for less than 12 hours. The findings suggest that this new approach could increase the availability of donor organs by reducing geographical limitations on donors and recipients, and enabling organs to be transported over longer distances to recipients further away than previously viable. Currently, about 200 adults are waiting for a lung transplant in Canada, and over 1500 in the USA [1]. About a quarter of those on the waiting list will die before they receive a transplant. Lung transplantation requires the donor organ to be stored and transported from the donor to the recipient. Traditionally, donor lungs have been flushed and preserved at cold temperatures to reduce tissue decomposition during transport. But the generally accepted maximum time from when an organ is removed from the donor, cooled, and then transplanted into the recipient is 6 to 8 hours. The recent development and use of EVLP around the world has completely altered the basic theory of lung preservation from slowing tissue death to preserving life, in order to allow and enhance recovery. This new technique involves continuously perfusing or pumping a bloodless solution containing oxygen, proteins, and nutrients into donor lungs to give doctors the opportunity to protect, assess and treat the lungs while they are outside the body and make them suitable for transplantation. "We have been using EVLP as standard practice to assess high-risk donor lungs for the last decade and almost 300 patients have benefited from this technology at our centre to date", explains lead author Dr Marcelo Cypel, Thoracic Surgeon at Toronto General Hospital, University of Toronto, Toronto, Canada. "Donor lungs are transported cold to the hospital where they are warmed, evaluated and then cooled again until they are transplanted into the recipient. Because assessing the lungs using EVLP takes at least 4 hours, total preservation times have regularly exceeded 8 hours."[2] Intrigued by the technique's possibilities for extending overall preservation times, Cypel and his team retrospectively examined data on the outcomes of 906 patients (aged 18 or older) who received a lung transplant at Toronto General Hospital between 2006 and 2015. They compared patients who had received a lung that had been preserved (i.e. the sum of cold preservation and normal temperature EVLP) for more than 12 hours (97 patients; 95% donor organs underwent EVLP) with those who were given lungs preserved for less than 12 hours (809; 5% underwent EVLP). They found that despite the use of higher-risk lungs in the more than 12-hour group, the average length of time recipients spent in the intensive care unit and in hospital post-transplant were similar in both groups (table 2). Additionally, the life-threatening complication of immediate graft dysfunction and survival at 1 year did not differ between the two groups (figure 2). Further analysis also showed that cold preservation and EVLP time did not affect survival (table 4). Older recipient age was the only factor that was linked with reduced survival. "It is important to remember that the lungs preserved for more than 12 hours using EVLP started out as more injured lungs. In fact, many of them might have been turned down for transplantation in the past. That they performed similar to conventional lungs with shorter preservation times suggests EVLP provides additional benefit over cold preservation", says first author Dr Jonathan Yeung, also from Toronto General Hospital [2]. The authors point to several limitations including the study's retrospective nature and the fact that it was conducted at a single institution. Additionally, the maximum safe preservation time for human lung transplantation remains unknown. They say that clinical trials are now needed to understand the optimum combination of cold and normal temperature EVLP lung preservation methods. According to Dr Cypel, "At a time when there is a critical shortage of lungs available for transplantation, combining cold preservation and EVLP will hopefully make a lot more donor lungs available for successful transplantation. This approach has allowed our Toronto-based programme to essentially abolish any geographical boundaries to donor lung retrieval in North America. The safe extension of preservation time not only has the potential to give clinicians extra time to evaluate, but also to treat and recondition donated organs that would otherwise not be used. Extra preservation time also allows additional flexibility in planning recipient surgery and gives more time to transport the donor lung from the EVLP site to the recipients' operating room."[2] Jonathan C Yeung (JCY), Thorsten Krueger (TK), Shaf Keshavjee (SK), and Marcelo Cypel (MC) conceived the study and contributed to its design. All authors participated in the acquisition, analysis, and interpretation of data. JCY, Thomas K Waddell (TKW), and MC contributed to the statistical analysis. JCY, SK, and MC contributed to drafting the paper. MC, TKW, and SK are co-founders of Perfusix Canada, XOR Labs Toronto, and consultants for Lung Bioengineering, United Therapeutics. [2] Quotes direct from authors and cannot be found in text of Article. IF YOU WISH TO PROVIDE A LINK TO THIS PAPER FOR YOUR READERS, PLEASE USE THE FOLLOWING, WHICH WILL GO LIVE AT THE TIME THE EMBARGO LIFTS: http://www.thelancet.com/journals/lanres/article/PIIS2213-2600(16)30323-X/abstract


VANCOUVER, BRITISH COLUMBIA--(Marketwired - Oct. 31, 2016) - Trevali Mining Corporation ("Trevali" or the "Company") (TSX:TV)(LMA:TV)(OTCQX:TREVF)(FRANKFURT:4TI) announces results from three diamond drill holes that are part of the ongoing 2016 underground exploration drill program at its Santander Zinc Mine in Peru. The aim of the current phase of drilling is to extend the down-dip expression of the Magistral Central (MC) zone, to test between Magistral North (MN) and MC zones in addition to test for the potential presence of hanging-wall hosted "Oyon" mineralization. Drill hole MC-181 targeted the area between MC and MN at the 4370 level where geological modeling indicted the presence of a high priority target. The sub-horizontal drill hole intersected 3.15 metres of high-grade Pb-Ag-Zn mineralization followed by 3.8 metres of high-grade Zn-Ag mineralization at approximately 94 metres downhole for an aggregate true thickness of 6.95 metres (Table 1). This exceptionally high-grade replacement mineralization remains open for expansion and step-out drilling is in progress (Figure 1 & 2). In detail, the mineralization is proximal to existing mine workings (just 95 metres from the main 4370 level) and readily accessible. Approximately 200 metres below MC-181, two drill holes (MC-179 and MC-180) intercepted new, narrow polymetallic mineralization which effectively links the Magistral bodies at these levels, and co-insides with the upper portion of the 2011 downhole geophysical anomaly that extends to depth (Figure 1 and see News Release dated October 24, 2011 for details). To view "Figure 1: 3D view (longitutinal cross-section) looking west showing Magistral Deposits with recent drill hole intercepts," and "Figure 2: Cross-section, looking north illustrating geometry of the high grade mineralization intersected at MC-181 and MC-171 (previously reported) at the Santander Zinc Mine," please visit the following link: http://media3.marketwire.com/docs/trevali_mining_oct31_fig01_02.pdf EurGeol Dr. Mark D. Cruise, Trevali's President and CEO and Daniel Marinov, P.Geo, Trevali's VP Exploration, are qualified persons as defined by NI 43-101, have supervised the preparation of the scientific and technical information that forms the basis for this news release. Mr. Marinov is responsible for all aspects of the work, including the quality control/quality assurance programs. Dr. Cruise is not independent of the Company, as he is an officer, director and shareholder. Mr. Marinov is not independent of the Company as he is an officer and shareholder. Drill core samples were processed and assayed in the Santander mine onsite laboratory. Zinc, lead and silver, assays were obtained by Aqua-Regia dissolution followed by Atomic Absorption measurement. Values of lead and zinc over 15% are assayed by volumetric method. Analytical accuracy and precision are monitored by the analysis of reagent blanks, reference material and replicate samples. Quality control is further assured by the use of international and in-house standards. Blind certified reference material is inserted at regular intervals into the sample sequence by Trevali personnel in order to independently assess analytical accuracy. The onsite laboratory is outsourced and managed by SGS-Peru personnel. SGS-Peru's quality system complies with the requirements for the International Standards ISO 9001:2000 and ISO 17025: 1999. Finally, representative blind duplicate samples are routinely forwarded to an ISO compliant third party laboratory for external quality control. Trevali is a zinc-focused, base metals mining company with two commercially producing operations. The Company is actively producing zinc and lead-silver concentrates from its 2,000-tonne-per-day Santander mine in Peru and its 3,000-tonne-per-day Caribou mine in the Bathurst Mining Camp of northern New Brunswick. Trevali also owns the Halfmile and Stratmat base metal deposits, located in New Brunswick, that are currently undergoing a Preliminary Economic Assessment reviewing their potential development. The common shares of Trevali are listed on the TSX (symbol TV), the OTCQX (symbol TREVF), the Lima Stock Exchange (symbol TV), and the Frankfurt Exchange (symbol 4TI). For further details on Trevali, readers are referred to the Company's website (www.trevali.com) and to Canadian regulatory filings on SEDAR at www.sedar.com. On Behalf of the Board of Directors of TREVALI MINING CORPORATION This news release contains "forward-looking statements" within the meaning of the United States private securities litigation reform act of 1995 and "forward-looking information" within the meaning of applicable Canadian securities legislation. Statements containing forward-looking information express, as at the date of this news release, the Company's plans, estimates, forecasts, projections, expectations, or beliefs as to future events or results and the Company does not intend, and does not assume any obligation to, update such statements containing the forward-looking information. Such forward-looking statements and information include, but are not limited to statements as to: the intended use of proceeds in connection with the Offering, the accuracy of estimated mineral resources, anticipated results of future exploration, and forecast future metal prices, expectations that environmental, permitting, legal, title, taxation, socio-economic, political, marketing or other issues will not materially affect estimates of mineral resources. These statements reflect the Company's current views with respect to future events and are necessarily based upon a number of assumptions and estimates that, while considered reasonable by the Company, are inherently subject to significant business, economic, competitive, political and social uncertainties and contingencies. These statements reflect the Company's current views with respect to future events and are necessarily based upon a number of assumptions and estimates that, while considered reasonable by the company, are inherently subject to significant business, economic, competitive, political and social uncertainties and contingencies. Many factors, both known and unknown, could cause actual results, performance or achievements to be materially different from the results, performance or achievements that are or may be expressed or implied by such forward-looking statements contained in this news release and the company has made assumptions and estimates based on or related to many of these factors. Such factors include, without limitation: fluctuations in spot and forward markets for silver, zinc, base metals and certain other commodities (such as natural gas, fuel oil and electricity); fluctuations in currency markets (such as the Canadian dollar and Peruvian sol versus the U.S. dollar); risks related to the technological and operational nature of the Company's business; changes in national and local government, legislation, taxation, controls or regulations and political or economic developments in Canada, the United States, Peru or other countries where the Company may carry on business in the future; risks and hazards associated with the business of mineral exploration, development and mining (including environmental hazards, industrial accidents, unusual or unexpected geological or structural formations, pressures, cave-ins and flooding); risks relating to the credit worthiness or financial condition of suppliers, refiners and other parties with whom the Company does business; inadequate insurance, or inability to obtain insurance, to cover these risks and hazards; employee relations; relationships with and claims by local communities and indigenous populations; availability and increasing costs associated with mining inputs and labour; the speculative nature of mineral exploration and development, including the risks of obtaining necessary licenses and permits and the presence of laws and regulations that may impose restrictions on mining, diminishing quantities or grades of mineral resources as properties are mined; global financial conditions; business opportunities that may be presented to, or pursued by, the Company; the Company's ability to complete and successfully integrate acquisitions and to mitigate other business combination risks; challenges to, or difficulty in maintaining, the Company's title to properties and continued ownership thereof; the actual results of current exploration activities, conclusions of economic evaluations, and changes in project parameters to deal with unanticipated economic or other factors; and increased competition in the mining industry for properties, equipment, qualified personnel, and their costs. Investors are cautioned against attributing undue certainty or reliance on forward-looking statements. Although the Company has attempted to identify important factors that could cause actual results to differ materially, there may be other factors that cause results not to be as anticipated, estimated, described or intended. The Company does not intend, and does not assume any obligation, to update these forward-looking statements or information to reflect changes in assumptions or changes in circumstances or any other events affecting such statements or information, other than as required by applicable law. Trevali's production plan at the Caribou Mine is based only on measured, indicated and inferred resources, and not mineral reserves, and does not have demonstrated economic viability. Trevali's production plan at the Santander Mine is based only on indicated and inferred mineral resources, and not mineral reserves, and does not have demonstrated economic viability. Inferred mineral resources are considered too speculative geologically to have the economic considerations applied to them that would enable them to be categorized as mineral reserves, and there is therefore no certainty that the conclusions of the production plans and Preliminary Economic Assessment (PEA) will be realized. Additionally, where Trevali discusses exploration/expansion potential, any potential quantity and grade is conceptual in nature and there has been insufficient exploration to define a mineral resource and it is uncertain if further exploration will result in the target being delineated as a mineral resource. We advise US investors that while the terms "measured resources", "indicated resources" and "inferred resources" are recognized and required by Canadian regulations, the US Securities and Exchange Commission does not recognize these terms. US investors are cautioned not to assume that any part or all of the material in these categories will ever be converted into reserves. This news release does not constitute an offer to sell or a solicitation of an offer to buy any of the securities in the United States. The securities described herein have not been and will not be registered under the United States Securities Act of 1933, as amended, or the securities laws of any state and may not be offered or sold within the United States, absent such registration or an applicable exemption from such registration requirements. The TSX has not approved or disapproved of the contents of this news release.


NEW YORK, Nov. 18, 2016 (GLOBE NEWSWIRE) -- Motif Bio plc (Motif)(NASDAQ:MTFB), a clinical stage biopharmaceutical company specializing in developing novel antibiotics, today announced the pricing of its initial public offering in the United States of approximately 2.44 million American Depositary Shares (ADSs) with 50% warrant coverage. Each ADS, representing 20 ordinary shares of Motif, and warrant to purchase 0.5 ADSs are being offered at an offering price to the public of $6.98 per ADS and warrant combination.  Motif has granted the underwriters a 30-day option to purchase up to an additional 292,618 ADSs and/or 146,309 warrants to cover over-allotments, if any, in the U.S. offering. Each full ADS warrant will have a per ADS exercise price of $8.03.  The ADS warrants are exercisable immediately and have a term of five years. The ADSs and warrants are scheduled to begin trading on The NASDAQ Capital Market on November 18, 2016 under the symbols "MTFB" and "MTFBW," respectively. H.C. Wainwright & Co. is acting as the sole book-running manager for the U.S. offering. Motif is also conducting a concurrent placement in Europe of 22.9 million ordinary shares with 50% warrant coverage. Each ordinary share and warrant to purchase 0.5 of an ordinary share are being offered at an offering price of 28 pence per ordinary share and warrant combination. Each full ordinary share warrant will have a per ordinary share exercise price of 32.2 pence.  The ordinary share warrants are exercisable immediately and will have a term of five years.  Motif’s ordinary shares trade on the AIM market of the London Stock Exchange under the ticker symbol “MTFB.” The Company’s placing agents have received signed placing letters committing to purchase the ordinary shares and warrants. Zeus Capital Limited, Northland Capital Partners and MC Services are acting as the placing agents in the European placement. The U.S. offering is expected to close on or about November 23, 2016, subject to customary closing conditions and the closing of the concurrent European placement. The aggregate gross proceeds to Motif from the U.S. offering and concurrent European placement are expected to be approximately $25.0 million before deducting underwriting discounts and commissions, placing agent commissions and other estimated offering expenses. The Company intends to use the net proceeds from these offerings, together with cash and cash equivalents on hand, (i) to fund the expenses to be incurred in conducting the two Phase 3 clinical trials of iclaprim for the treatment of ABSSSI, including the completion of our REVIVE-1 trial; and (ii) for working capital, general and administrative expenses, research and development expenses, and other general corporate purposes. While the Board believes that, along with the Company’s existing cash and cash equivalents, the net proceeds from the U.S. offering and concurrent European placement will provide sufficient capital to enable the Company to complete the REVIVE-1 trial, the Company will require additional funds to complete the REVIVE-2 trial and plans to raise the additional capital through public or private financings and/or other partnering opportunities. A registration statement relating to these securities was declared effective by the Securities and Exchange Commission on November 17, 2016.  A final prospectus relating to this offering will be filed with the U.S. Securities and Exchange Commission. This offering is being made only by means of a prospectus. Copies of the prospectus relating to this offering may be obtained by contacting H.C. Wainwright & Co., 430 Park Avenue, New York, NY 10022, telephone: 212-356-0500, or e-mail: placements@hcwco.com. Investors may also obtain these documents at no cost by visiting the SEC's website at http://www.sec.gov. This press release shall not constitute an offer to sell or a solicitation of an offer to buy, nor shall there be any sale of these securities in any state or jurisdiction in which such an offer, solicitation or sale would be unlawful prior to registration or qualification under the securities laws of any such state or jurisdiction. About Motif Bio Motif Bio is a clinical-stage biopharmaceutical company, engaged in the research and development of novel antibiotics designed to be effective against serious and life-threatening infections in hospitalized patients caused by multi-drug resistant bacteria. Our lead product candidate, iclaprim, is being developed for the treatment of acute bacterial skin and skin structure infections (ABSSSI) and hospital acquired bacterial pneumonia (HABP), including ventilator associated bacterial pneumonia (VABP), which is often caused by MRSA (methicillin resistant Staphylococcus aureus). We are currently enrolling and dosing patients in two global Phase 3 clinical trials (REVIVE-1 and REVIVE-2) with an intravenous formulation of iclaprim, for the treatment of ABSSSI.  Data readout for REVIVE-1 is expected in the second quarter of 2017 and REVIVE-2 is on track for data readout in the second half of 2017. Forward Looking Statements This press release contains forward-looking statements. Words such as "expect," "believe," "intend," "plan," "continue," "may," "will," "anticipate," and similar expressions are intended to identify forward-looking statements. These forward-looking statements involve certain risks and uncertainties, including, among others, risks impacting the ability of Motif Bio plc to complete any public offering of its securities because of general market conditions or other factors and risks that could cause the results to differ materially from those expected by the management of Motif Bio plc. More detailed information about the risks and uncertainties affecting Motif Bio plc is contained under the heading "Risk Factors" in Motif Bio plc's registration statement on Form F-1 filed with the SEC, which is available on the SEC's web site, www.sec.gov. Motif Bio plc undertakes no obligation to update or revise any forward-looking statements.


News Article | November 7, 2016
Site: www.businesswire.com

DUBAI, United Arab Emirates--(BUSINESS WIRE)--Expway and Bittium offer the first ever LTE Broadcast enabled ruggedized mobile devices which enable uninterrupted, secure and scalable MC-PTT, MC-Video, and MC-Data.


News Article | February 15, 2017
Site: www.prweb.com

Welcome to the 2017 New York Pet Fashion Show Presented by TropiClean. This year’s theme is “Global Couture for Animal Rescue”, and features World Fashion to Save our Animals and Honor our Planet. The show is a celebration of our love for animal rescue which helps save animals' lives, and will benefit The Mayor's Alliance for NYC's Animals and their 150 participating shelters and rescue groups. The NY Pet Fashion Show is honored to have Merck Animal Health, Tomlyn, and Hamptons Pet magazine as this year’s Best in Show sponsors.. This year's host and MC is Celebrity Pet Expert Harrison Forbes, and the Gold Paw awards will feature Beth Shak-Levenathal as the 2017 “Humanitarian” award winner. A special appearance with the “Best of Broadway” featuring Hamilton, Cats, Phantom of the Opera, Lion King, Wicked, Kinky Boots, Chicago, Aladdin, and more. The models and designers will feature Couture Fashion inspired by their favorite Designer, Country, Culture, Ethnicity, or City. Fashions from Italy, France, Thailand, Egypt, USA, United Kingdom, India, South Korea, Puerto Rico, Paraguay, Mexico, and more. Dress to Impress! The 14th annual New York Pet Fashion Show Presented by TropiClean kicks off Westminster Weekend and Fashion Week. The 2017 theme is “Global Couture for Animal Rescue”, and is being held on Feb 9th 2017 at the Hotel Pennsylvania in NYC. Celebrities and animal rescue stars will be walking the runway. The runway will feature Rachel Grant, a “Bond Girl” from Die Another Day, and Bravo’s Tour Group, along with actress Naomie Olindo from Bravo's "Southern Charm, and Urban Animals Robbyne Kamil. The New York Pet Fashion Show Presented by TropiClean also features the Gold Paw Awards ceremony, which celebrates amazing people, pets, and volunteers. The NY Pet Fashion Show is the largest pet fashion and animal rescue event in the United States, where celebrities, rescues, models and designers strut their stuff on the runway. http://www.NYPetFashionShow.com Chairman, Gregg Oehler invites the media to cover the event of the season, which is the Super Bowl of fashion and rescue. The show averages 600-800 people in attendance, with over 100 media covering the event. This event will be filmed for TV, and features three runway shows directed by world renowned pet fashion designer and the shows Co-Chair and creative director Ada Nieves. The show will feature must see top pet fashion designers from across the World showcasing their couture collections! New York City is the Capital of the world in media, fashion, finance, and is now the dog, cat, and animal rescue capital of the world. See the top pet designers from across the globe and their creations, with cats, dogs, chickens, rats, and a bearded lizard on the runway. Media: To be placed on the VIP Media Guest list or contact for interviews: Gregg(at)OehlerMedia(dot)com or call 203-595-5452 To Purchase Your NYPFS Ticket(s) click the link below http://nypetfashionshow.com/index.php?id=buy-tickets Tickets: To the public $50 in advance/$60 at the door Address: 401 Seventh Ave, 33rd Street, New York, NY. 10001 Where: Hotel Pennsylvania 18th Fl. Penntop Ballroom When: Thursday February 9th, 2017 from 6 p.m. - 11 Info: For more info call 646-202-3807 2017 New York Pet Fashion Presented by TropiClean Event Sponsors- TropiClean, Merck Animal Health, Tomlyn, Hamptons Pet, and ,PetMediaPR Gregg R. Oehler-Chairman, NY Pet Fashion Show, Oehler Media Inc W: http://www.NYPetFashionShow.com P: 203- 595- 5452 // C: 646-202-3807 An Oehler Media Production


INGLEWOOD, CA--(Marketwired - Nov 1, 2016) - Green Cures & Botanical Distribution ( : GRCU) will be launching a line of CBD and THC sodas in four flavors devoted to Digital Underground, hip-hop pioneers. With flavors dedicated to members of the band and some of the bands legendary song titles nationwide. This deal with Purple Haze Properties is expected to launch up to four new flavors each year and will be sold to supermarkets without cannabis and dispensaries with cannabis in every authorized state. The Digital Underground soda line will also have distribution at concerts and venues that the band will be playing. Currently, the group is working on a new album and will be touring on the heels of the new Tupac Shakur Movie, "All Eyez on me," a biopic film that focuses on Tupac's life and career, including his time at Death Row Records and his intense involvement with rivalry East and West Coast Rappers, culminating in his murder at the age of 25 in 1996. This film will be coming out in early 2017 by Morgan Creek Productions. Tupac was a roadie, a legendary songwriter, hip-hop artist, a back-up dancer and MC for Digital Underground. Money B, a member of Digital Underground, was asked to play himself in this new release. Those interested in the trailer of the movie visit: http://alleyezonmethemovie.com. Money B stated today, "I can't wait for these great products to come out by GRCU. The soda category has been the hot trend in CBD and cannabis infused drinks. A few beverages have won the High Times Cannabis Cup this year, and GRCU is making their mark in this industry." "Soda is a great way to mask the CBD and THC flavor without compromising quality. By offering another great celebrity brand to Iconic Beverages, we can make really creative drinks the fans will love," states, Andrew Pitsicalis, CEO of Purple Haze properties and GRCU Spokesperson. We look forward to these great beverages in a store near you next year. Please visit www.purplehazeproperties.com for Release Dates, Flavor Previews, Events and Announcements for GRCU as well as www.originalhollywoodhemp.com for additional information and product sales. Purple Haze Properties, LLC was founded through a partnership between Andrew Pitsicalis and Leon Hendrix, blood brother to the legendary guitar icon. Purple Haze Properties, LLC is the premiere source for Jimi Hendrix cannabis related products. Purple Haze Properties is a premiere celebrity licensing company for the Cannabis Industry. We represent, Cannabis Licensing for many celebrities including, Jimi Hendrix, Motorhead, Digital Underground, 311, Anthrax, Charlo Greene, The Emperor of Hemp, Jack Herer, Baby Bash and others. Purple Haze Properties participates in all aspects of the cannabis industry including Licensing, Social, Medical, Music and Entertainment areas. Check us out at www.purplehazeproperties.com or www.facebook.com/purplehazeproperties. Green Cures & Botanical Distribution Inc. (GRCU) develops, produces & distributes premium hemp based products in the following categories: Medical, Skin Care, Beauty, Fashion and lifestyle goods branded under the Original Hollywood Hemp™ Brand. Green Cures & Botanical Distribution Inc. (GRCU) also develops beverages branded under the Iconic Beverages ™ and others. Green Cures & Botanical Distribution Inc. is a revenue-generating company that wholesales and retails hemp-infused nutritional, botanical, sports, and body care products. The company is currently Web-based and focuses on online retailing. Green Cures & Botanical Distribution Inc. operates a diverse portfolio of products and services within the botanical and cannabis industry, as permitted by law. From concept to production and distribution, Green Cures & Botanical Distribution Inc. is continuously creating and introducing products that promote a healthy life. This press release contains forward-looking statements within the meaning of Section 27A of the Securities Act of 1933, as amended, and Section 21E of the Securities Exchange Act of 1934, as amended. In some cases, you can identify forward-looking statements by the following words: "anticipate," "believe," "continue," "could," "estimate," "expect," "intend," "may," "ongoing," "plan," "potential," "predict," "project," "should," "will," "would," or the negative of these terms or other comparable terminology, although not all forward-looking statements contain these words. Forward-looking statements are not a guarantee of future performance or results, and will not necessarily be accurate indications of the times at, or by, which such performance or results will be achieved. Forward-looking statements are based on information available at the time the statements are made and involve known and unknown risks, uncertainty and other factors that may cause our results, levels of activity, performance or achievements to be materially different from the information expressed or implied by the forward-looking statements in this press release. This press release should be considered in light of all filings of the Company that are contained in the Edgar Archives of the Securities and Exchange Commission at www.sec.gov.

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