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TSD-ML11 1 x 1 Mic/Line Preamp with EQ & Mute TSD-ML11 LIMITER 1x1 Mic/Line Preamp INPUT PEAK LIMITER SIGNAL INPUT PEAK POWER TSD-ML11 1x1 Mic/Line Preamp – + INPUT – – + INPUT + SIGNAL OUTPUT 1601 Jack McKay Blvd. • Ennis, TX 75119 • 800.876.3333 • AtlasSound.com – POWER + OUTPUT 1601 Jack McKay Blvd. • Ennis, TX 75119 • 800.876.3333 • AtlasSound.com 1 2 3 ©2011 Atlas Sound L.P. All rights reserved. Atlas Sound is a trademark of Atlas Sound L.P. All other trademarks are the property of their respective owners. INPUT 4 5 6 INPUT 4 LINE OUTPUT 5 LINE 250Hz 6 OFF MIC INPUT MIC OUTPUT ON 250Hz LINE LINE OFF MIC MIC ON 7 PHANTOM 2 OFF OFF OFF OFF ON ON ON LOW CUT 3 OFF 125Hz/12dB ON ON PHANTOM LOW CUT – + 24VDC 100mA – + 24VDC 100mA G – + INPUT G – + INPUT 8 9 EQ CUT 12dB 1/3 OCT 125Hz/12dB ON 1 OFF INPUT ON ON G – + OUTPUT G – + OUTPUT INPUT INPUT MUTE ON 1kHz 2.5kHz 7 8 OFF OFF EQ CUT 12dB 1/3 OCT ON ON 1kHz 2.5kHz OFF OFF ON ON 1 2 3 4 5 6 7 8 9 ON 1 2 3 4 5 6 7 8 9 ON INPUT INPUT MUTE ON 6.3kHz 9 OFF ON 6.3kHz OFF ON 0dB –dB OUTPUT LIMITER 0dB –dB OUTPUT LIMITER TSD-ML11 Features • High Quality Microphone Preamp • Mic/Line I/O via Removable Phoenix Style Connector • 125Hz/12dB Low Cut Filter • Phantom Power • Four (4) Preset Frequency Notch Filters For Tone and Feedback Control • Variable Output Limiter/Compressor • Mic Mute/Mic On Via Contact Closure • Input Signal & Peak Indicator LEDs • High Gain Output for Long Distance Applications • Separate Ground Connections • Cable Management Systems • Multiple Mounting Configurations • Removable Phoenix Style I/O Connectors • Security Covers Included for Front Panel Level Controls Atlas Time Saving Devices New Atlas Sound Time Saving Devices are designed to solve some of the common audio system problems in the easiest and in the most efficient and cost-effective way. Utilizing the highest quality components and inspired by real world installation issues, the Atlas Sound TSDs include mixers, amplifiers, preamps, and other distribution and control devices to simplify your connections, provide specific application needs, and add flexibility in expansion and changes that prevent expensive system overhauls. The Time Saving Devices are loaded with features making it versatile and user friendly. The TSD design mandate was to make the installation quick and reliable, saving you valuable time and money. Each model is housed in a compact 1.5" x 4" x 2.75" footprint that allows for placement almost anywhere. Common features include removable individual I/O connectors for quick and precise wiring, separate ground terminals for all I/Os assuring secured connections, and removable level controls that can be replaced with a tamper-resistant security cover. Description The TSD-ML11 is a high headroom, low noise mic preamp that allows your 'money channel' to have the highest performance possible. Use as a standalone preamp or in conference room systems, board meetings, courtrooms, council chambers, and public forums system that need mute and push to talk microphone functions. The TSD-ML11 features switchable phantom power for powering condenser microphones and has four EQ cut filters centered at common frequencies to eliminate feedback and control voice shaping. The variable mic preamp helps further eliminate noise for quiet operation as well as reduces unwanted coloration and changes in tonality and a 12dB @ 125Hz high pass filter is available on each mic input to minimize bass rumble and low frequency feedback in the system and ensure focus and clarity stays on voice sound spectrum. An output limiter/compressor is also included for system protection to avoid clipping and eliminate distortion. Mic Mute and Mic On control allows microphones in the system to be push-to-talk enabled or muted for speaker priority. The TSD-ML11 is highly efficient and meets Energy Star™ standards as one 24V DC power supply (available separately) can power multiple Atlas Time Saving Devices. Installation • Meets Energy Star™ Standards • Churches • Compact Chassis Design • Restaurants Audio Applications • Sports Bars • Mic Preamp • Line Preamp • Line Drive • Schools • Industrial Facilities • House of Worship • Choirs • Remote Mic Control 1601 JACK MCKAY BLVD. ENNIS, TEXAS 75119 U.S.A. TELEPHONE: (800) 876-3333 FAX (800) 765-3435 AtlasSound.com 1/3 ©2011 Atlas Sound L.P. All rights reserved. Atlas Sound is a trademark of Atlas Sound L.P. All other trademarks are the property of their respective owners. Specifications Description 1 x 1 Mic/Line Preamp Chassis Color Chassis Material Black Aluminum Inputs Type Connection Impedance Phantom Low Cut Filter Line Input Level Mic Input Level Qty 1, Balanced Mic or Line Selectable Removable 3 Position Captive Screw 3.5mm Spacing 1.2KΩ Balanced, 600Ω Unbalanced 24VDC Defeatable 125Hz/12dB Defeatable 1V (0dBV) Balanced 2V (+6dBV) Balanced Output, Maximum Input 8V (+15dBV) 46dB Gain 10mV (-40dB) Balanced = 2V (+6dBV) Balanced Output, Maximum Input 40mV (-27dBV) Outputs Type Connection Impedance Line Output Level Mic Output Level Qty 1, Balanced Mic or Line Selectable Removable 3 Position Captive Screw 3.5mm Spacing 300Ω Balanced, 150Ω Unbalanced 1V (0dBV) In = 2V (+6dBV), Maximum Output 6V (15dBV) 10mV (-40dBV) In = 80mV (-21dBV), Maximum Output 300mV (-10dBV) Controls Input Level Control Output Level Control Output Limiter Mic Mute Mic On Dip Switch Function Qty 1, Removable Knob with Security Cover Qty 1, Removable Knob with Security Cover Threshold Adjust From +18dB - No Output. Hard Knee Limit Contact Closure, Removable 2 Position Captive Screw 3.5mm Spacing Contact Closure, Removable 2 Position Captive Screw 3.5mm Spacing • Input 1 On/Off • Phantom Power 24VDC • Input Mic/Line Select • Input 125Hz/12dB High Pass Filter • Output Qty 1, Mic/Line Select • EQ, 12dB Cut Filters 250Hz, 1KHz, 2.5KHz, 6.3KHz Indicators Input Signal Input Peak Limiter Power Green, 25mV On Threshold Red, -3dB Before Clip Red, Output Limiter Activation Blue Electrical Frequency Response THD Output Limiter Signal to Noise 20Hz - 20kHz (±1dB) .06% & 1kHz Threshold Adjust 50mV to No Limiter Line to Line: 85dBV Mic to Line: 70dBV Line to Mic: 55dBV Mic to Mic: 55dBV Mounting Velcro Brackets TSD-RMK Cable Retainer Industrial-Grade, Included Qty 2, Included Optional 1 RU Rack Mount Panel (Supports 4 TSD Units) Qty 2, Included Power Requirements Voltage Power Consumption Power Consumption 24VDC 100mA 2.4W 1601 JACK MCKAY BLVD. ENNIS, TEXAS 75119 U.S.A. TELEPHONE: (800) 876-3333 FAX (800) 765-3435 AtlasSound.com 2/3 Dimensions H 1.5" (39mm) x W 4" (101mm) x D 2.75" (70mm) Weight 6oz, .165kg Accessories TSD 24VDC Power Supplies TSD-RMK 1 RU Rack Panel Safety and Certifications External Power Supply cUL, CE, RoHS, TUV Block Diagram DIP SWITCH FUNCTION SET 1 2 INPUT 3 4 5 6 7 ON PHANTOM LOW CUT INPUT OUTPUT 250Hz 1kHz 2.5kHz 6.3kHz OFF OFF LINE LINE OFF OFF OFF OFF ON ON ON MIC MIC ON ON ON ON 1kHz CUT 12dB/Q5 2.5kHz CUT 12dB/Q5 3 4 5 6 7 8 9 ©2011 Atlas Sound L.P. All rights reserved. Atlas Sound is a trademark of Atlas Sound L.P. All other trademarks are the property of their respective owners. 250Hz CUT 12dB/Q5 INPUT 9 OFF 1 2 PHANTOM 8 EQ CUT 12dB 1/3 OCT 125Hz/12dB 6.3kHz CUT 12dB/Q5 DC 24V INPUT MIC/LINE SELECT MINI PHOENIX INPUT LEVEL + – + LOW CUT 125Hz – OUTPUT LIMITER G OUTPUT MIC/LINE SELECT OUTPUT LEVEL MIC/LINE OUTPUT G OUTPUT MUTE INPUT PEAK OUTPUT ON DC 24V OUTPUT LIMIT SIGNAL OUTPUT ON BYPASS POWER LED 1601 JACK MCKAY BLVD. ENNIS, TEXAS 75119 U.S.A. TELEPHONE: (800) 876-3333 FAX (800) 765-3435 AtlasSound.com 3/3
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Parkour Level 2 test
Students must demonstrate mastery of the parkour level 2 material before moving to parkour level 3 classes. In order to do so, they must take a standard parkour level 2 test. The test is pass/fail, and is scaled so that the most difficult movements on the test are first. Failure to complete any element of the test ends the test.
Strength qualifier
In order to qualify to take the test, students must demonstrate the following strength movements with perfect form:
Handstand on wall (nose to wall) 60 seconds
- Straight body
- Hands close to wall
Tuck planche
Tuck planche 10 seconds
- Hips in line with shoulders
Knees to elbows
Knees to elbows - 10 reps
- From a dead hang
- Controlled movement (no yanking up)
- Knees must come to elbows
Pull ups
5 pull ups
- One smooth motion
- Chest to bar
- Shoulders back/neutral
Decline push ups
10 decline push ups with feet on knee-height obstacle
- Straight body
- Elbows around 45 degrees
- Full range of motion
One-leg squats
5 one-leg (shrimp, first progression) squats each leg
Video forthcoming. In the meantime, see Demon Drills demo:
Skills test
180 cat, climb up, drop, roll
180 cat between jugs or head-height obstacles at 1.5BL, climb up, top out, drop and roll. Repeat.
(Level 2 climbup)
Wall pass
Wall pass 1.75x your height, symmetrical climb up and top out. Repeat.
Lache to grab level bar at 1.25x your body length. Repeat.
Dynamic jumps
Plyo/stride: From standing start, 2 strides to precision for 4BL distance. Repeat.
Kong to cat
Kong waist-high obstacle to cat on head height obstacle at 1BL. Repeat.
High commitment vaults
Speed/dash/hurdle over waist high obstacle, both sides.

subset: commoncrawl


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From Our Editors
Step into a whole new world of style when you browse the apparel offerings at Bella B's Boutique in Lee'S Summit. Whether you need a new pair of jeans, a new wardrobe size, or just a new look altogether, this store is every woman's fashion dream come true. Hats, gloves, and scarves galore are available at Bella B's Boutique.
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Chronic oxidative stress has been associated with genomic instability following exposure to ionizing radiation. However, results showing direct causal linkages between specific ROS (reactive oxygen species) and the ionizing radiation-induced mutator phenotype are lacking. The present study demonstrates that ionizing radiation-induced genomically unstable cells (characterized by chromosomal instability and an increase in mutation and gene amplification frequencies) show a 3-fold increase in steady-state levels of hydrogen peroxide, but not superoxide. Furthermore, stable clones isolated from parallel studies showed significant increases in catalase and GPx (glutathione peroxidase) activity. Treatment of unstable cells with PEG-CAT (polyethylene glycol-conjugated catalase) reduced the mutation frequency and mutation rate in a dose-dependent fashion. In addition, inhibiting catalase activity in the stable clones using AT (3-aminotriazole) increased mutation frequency and rate. These results clearly demonstrate the causal relationship between chronic oxidative stress mediated by hydrogen peroxide and the mutator phenotype that persists for many generations following exposure of mammalian cells to ionizing radiation.
For many years, it was thought that genetic changes caused by radiation were only due to DNA damage derived from the incident free radical species produced at the time of exposure. However, it was discovered that the number of surviving cells exhibiting increased delayed mutation frequencies following ionizing radiation is greater than would be predicted if only the cells exposed at the time of irradiation were involved [1,2]. In other words, the mutagenic effects of ionizing radiation were found to be trans-generational. These persistent trans-generational mutagenic effects, apparently resulting from a mutator phenotype, have collectively been called non-targeted or bystander effects of radiation, but the mechanisms governing these phenomena are not well understood [1,2].
Chronic oxidative stress mediated by continuous exposure to H2O2 or 95% O2 has been shown to induce genomic instability and gene amplification in hamster fibroblasts [3]. Additional studies investigating the cause of ionizing radiation-induced genomic instability have suggested that persistent metabolic oxidative stress may be involved [4,5]. Hydroperoxides produced by metabolic processes have been suggested to contribute to persistent ionizing radiation-induced genomic instability [6,7] on the basis of data gathered using non-specific oxidation-sensitive probes. However, direct causal linkages between increased steady-state levels of specific ROS (reactive oxygen species), such as H2O2, and the mutator phenotype characterized by increased rates of delayed mutagenesis following exposure to ionizing radiation are lacking.
In the present study, three subsets of Chinese hamster ovary fibroblasts (GM10115 cells) were used to determine the involvement of H2O2 in the ionizing radiation-induced mutator phenotype: (i) the untreated parental GM10115 cell line, (ii) unstable GM10115 clones designated CS-9 and LS-12, and (iii) stable clones designated 114 and 118 [8]. We now show that the unstable clones not only exhibit increased mutation frequency and rates, but were also found to produce greater steady-state levels of H2O2 relative to the parental cell line and stable clones. The stable clones were found to have increased catalase and GPx (glutathione peroxidase) activities relative to the unstable clones. Furthermore, both increased mutation frequency and increased mutation rates in the unstable clones were suppressed by a cell-permeant H2O2-scavenging enzyme, PEG-CAT [PEG (polyethylene glycol)-conjugated catalase]. Mutation frequencies and rates could be increased in the stable clones treated with the catalase inhibitor AT (3-aminotriazole). These results provide unambiguous evidence showing that H2O2 mediates the persistent mutator phenotype in these mammalian cells and suggest that scavengers of H2O2 could be used to inhibit chronic indications of genomic instability persisting many generations after exposure to a range of cytotoxic treatments.
Cell culture
The Chinese hamster ovary parental cell line GM10115 was obtained from A.T.C.C. Cell stock flasks were grown in DMEM (Dulbecco's modified Eagle's medium) with high glucose and 1 mM sodium pyruvate (CellGrow), 10% fetal bovine serum (Hyclone), 0.2 mM L-proline and 1% L-glutamine (Gibco). The previously described [8] stable (114, 118) and unstable (CS-9, LS-12) clones were grown in the same media.
Mutation frequency assay
Cells (3×106) from an exponentially growing culture were seeded on to 100-mm-diameter dishes. DMSO was used to dissolve 6-thioguanine (Sigma) before it was added at a final concentration of 40 μM. The dishes were left undisturbed for 2–3 weeks until colonies with at least 50 cells appeared. The colonies were then fixed, stained and counted. The data were normalized to plating efficiency of identically treated cells in the absence of 6-thioguanine.
CAD (carbamoyl-P synthetase/aspartate transcarbamylase/dihydro-orotase) gene amplification assay
Amplification at the CAD gene locus was determined by plating 3.5×105 cells on a 100-mm-diameter dish. Medium containing 100 μM PALA (N-phosphonoacetyl-L-aspartate) [Developmental Therapeutics Program, NCI (National Cancer Institute)/NIH (National Institutes of Health)] was added and dishes were placed in a 37 °C incubator for 2 weeks. The colonies were then fixed, stained and counted. The number of colonies was normalized to the plating efficiency.
Oxidation of c-DCFH2 [5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate] as an indicator of steady-state levels of pro-oxidants
Cells (3.5×105) were seeded on a 60-mm-diameter dish in complete medium (three replicates for each clone) and grown for 2 days. On day 2, the cells were washed with PBS, following which 2 ml of PBS was added to each dish and the dishes were incubated with 10 μg/ml c-DCFH2 (Invitrogen), or the oxidation-insensitive analogue c-DCF [5-(and 6-)carboxy-2′-7′-dichlorofluorescein], for 15 min at 37 °C. The cells were then washed, trypsinized and centrifuged at 400 g for 5 min. The pellet was resuspended in 500 μl of PBS and filtered through a mesh to remove large aggregates. Live cells (10000; gated on forward and side scatter) were analysed for C-DCF fluorescence (FL1 channel, λexcitation=488 nm, λemission=530 nm, 30 nm bandpass) using a Beckton Dickinson FACScan. DMSO (0.1% v/v) and antimycin A (10 μM) dissolved in DMSO were used as negative and positive controls respectively. CellQuest Pro software was used for data analysis.
Oxidation of DHE (dihydroethidium) as an indicator of steady-state levels of superoxide
Cells (3.5×105) were seeded on 60-mm-diameter dishes (three replicates for each clone) and grown for 2 days in complete media. The cells were labelled, after washing the plates with PBS, by incubating at 37 °C for 40 min with 10 μM DHE (Invitrogen) and DMSO (0.1% v/v) in 2 ml PBS containing 5 mM pyruvate. The cells were then washed, trypsinized and centrifuged at 400 g for 5 min. The pellet was resuspended in 500 μl of PBS and filtered through a mesh to remove large aggregates. Live cells (10000), gated on forward and side scatter, were analysed for DHE fluorescence using a Beckton Dickinson FACScan (FL2 channel, λexcitation=488 nm, λemission=585 nm, 30 nm bandpass). DMSO (0.1% v/v) and antimycin A (10 μM) dissolved in DMSO were used as negative and positive controls, respectively. CellQuest Pro software was used for data analysis.
Extracellular hydrogen peroxide
The hydrogen peroxide assay was carried out as previously described [9]. Exponentially growing cells were washed with Phenol Red-free HBSS (Hanks balanced saline solution) three times. This was followed by the addition of glucose (final concentration 6.2 mM), Hepes (final concentration 1.0 mM), sodium bicarbonate (final concentration 6.0 mM), p-HPA (p-hydroxyphenylacetic acid, Sigma) (final concentration 1.6 mM) and HRP (horseradish peroxidase; final concentration 95 μg/ml). HBSS was used to bring the final volume to 1 ml. The cells were incubated at 37 °C for 30 min, during which time H2O2 accumulated in the extracellular medium. In the case of catalase samples, to verify that the signal was coming from H2O2, the dishes were incubated with catalase (1000 units) for 30 min prior to the addition of reaction mixture. The media was collected in a cuvette and the release of H2O2 was followed fluorometrically (Perkin Elmer LS50B) by measuring the p-HPA dimer formed at excitation and emission wavelengths of 320 and 408 nm respectively. Quantitation was accomplished using comparison to a standard curve obtained from genuine H2O2. The pH of the samples was checked following incubation and did not change significantly.
Isolation of mitochondria
Mitochondria were isolated from cells using density gradient centrifugation with a sucrose gradient in isolation buffer (0.25 M sucrose, 5 mM Hepes, 0.1 mM EDTA, pH 7.25) [10]. Exponentially-growing cells were harvested and centrifuged at 500 g. The pellet was resuspended in isolation buffer and homogenized using a Dounce homogenizer rinsed with dilute HCl followed by distilled water. Cell debris was separated by centrifugation at 1000 g for 10 min. The supernatant was collected from this step and centrifuged at 10000 g for 10 min. The mitochondrial pellet was resuspended in 500 μl of isolation buffer and used for further analysis. Protein was determined using the Bio-Rad protein assay protocol.
EPR analysis was carried out on isolated mitochondria using a Bruker EMX spectrometer. Mitochondrial protein samples (100 μg) were incubated with the DMPO (5,5-dimethyl-1-pyrroline N-oxide) spin trap (150 mM) for 2 min at room temperature (20–25 °C). This was followed by a 10 min incubation with 9 mM succinate (pH adjusted to 7.4) at 37 °C. To ascertain that the DMPO-OH EPR signal observed was indeed due to superoxide, CuZnSOD (Cu/Zn superoxide dismutase; 1000 units) was added 2 min prior to the addition of DMPO to suppress the EPR signal. The part of the signal that was suppressed by the enzyme was considered a true measure of superoxide and thus this portion was plotted. Spectra were collected using TM (transverse magnetic) cavity and a flat cell. Spectra were quantified by measuring the signal intensity of the two centre lines of DMPO-OH spectra (aN=14.9 G and aH=14.9 G) and averaging these values [11]. The instrument parameters used were: sweep width 80 G, power 40 mW, ν=9.79 GHz, scan rate 82 s, modulation amplitude 1 G, time constant 82 ms, number of scans=10.
GPx activity
To make cell homogenates, 50 mM potassium phosphate buffer (1:1 KH2PO4/K2PO4), pH 7.8, containing 1.34 mM DETAPAC (diethylenetriaminepenta-acetic acid), was added to cell pellets following one freeze/thaw cycle. GPx-1 activity was measured using cumene hydroperoxide (Sigma) as the substrate by monitoring the disappearance of NADPH at 340 nm as described previously. Data were normalized per mg of protein as determined by the Lowry protein assay [12,13].
Catalase assay
Catalase activity was determined by monitoring the disappearance of 10 mM H2O2 in reactions containing cell homogenate as previously described [14]. Activity was expressed in mκunits/mg of protein.
GSH measurements
The recycling assay of Anderson and Griffith [15,16] was used to determine glutathione content in cell homogenates and was normalized to protein content. GSSG was determined by incubating the sample with 2-vinylpyridine as previously described [16]. GSH and GSSG concentrations were reported in GSH equivalents using a standard curve.
SOD activity assay
The SOD activity assay was performed as previously described [17]. Inhibition of NBT (Nitro Blue Tetrazolium) reduction was used as a marker of SOD activity. Samples were run either in the presence of 5 mM cyanide (for MnSOD activity) or in the absence of cyanide (for total SOD activity). MnSOD activity was subtracted from the total, and the propagation of errors theory was used to obtain CuZnSOD activity.
PEG-CAT treatment
Exponentially growing cells (3×106) were seeded in 100 mm dishes, and 100 units/ml PEG-CAT (Sigma) (reconstituted in PBS) was added to each dish 2 h prior to the addition of 6-thioguanine. PEG was added at an equivalent concentration to the conjugated enzyme to control for any effects not specific to the enzyme activity.
Aminotriazole treatment
Cells were grown in complete media with 20 mM AT for 4 days. On the fourth day, cells were trypsinized, counted, and 3×106 cells were seeded in media containing 40 μM 6-thioguanine without AT. After 2 weeks, colonies were stained and counted. The effect of AT on the plating efficiency of cells was corrected for in the mutation frequency assay.
Mutation rate assay
Cells were selected in media containing HAT (CellGrow) (hypoxanthine/aminopterin/thymidine) for 3 weeks. Following this, cells were grown in complete medium without HAT and mutation frequency was assayed immediately following removal from HAT selection (day 0) as well as after 10–15 days of passage in the absence of HAT. Mutation frequency was divided by the number of days out of selection to determine mutation rate/106 cells per day. The mutation rates for comparable groups were determined on the exact same day [18].
Statistics were performed using one-way ANOVA for non-parametric measurements. Tukey post-hoc test was performed wherever appropriate. Results were considered significantly different if P≤0.05 unless otherwise mentioned. All statistical tests were done using GraphPad Prism software.
Clones used in this study were originally described by Limoli et al. [8]. Briefly, colonies derived from single cells surviving exposure to 10 Gy were expanded and analysed for multiple endpoints of genomic instability. To confirm past findings, and to verify that an unstable rather than a stable clone possessed a mutator phenotype, cells were analysed for mutation and gene amplification frequencies. Our data show that the unstable clone CS-9 exhibits higher (4- to 5-fold) mutation and gene amplification frequencies, at the hprt (hypoxanthine–guanine phosphoribosyltransferase) and CAD gene loci respectively, relative to the parental GM10115 and stable 114 clone (Figures 1A and 1B).
CS-9 cells show increased genomic instability
When steady-state levels of pro-oxidants were determined, the unstable clones (CS-9 and LS-12) demonstrated significantly increased c-DCFH2 oxidation relative to the parental and the stable clone 114 (Figure 2A). These changes in c-DCFH2 fluorescence were confirmed to be truly indicative of changes in steady-state pro-oxidant levels (and not changes in probe uptake, ester cleavage or efflux) because no significant changes in fluorescence were noted in co-cultures labelled with the oxidation-insensitive analogue, c-DCF (results not shown). Consistent with the c-DCFH2 results, when a specific measure of extracelluar H2O2 production was determined (catalase-inhibitable p-HPA oxidation), the unstable clones (CS-9 and LS-12) were found to produce significantly more H2O2 relative to the parental and stable 114 clone (Figure 2B). None of the unstable clones showed a consistent change in steady-state levels of superoxide in isolated mitochondria as determined by SOD-inhibitable EPR spectroscopy (Figure 2C), and only LS-12 showed a modest change in superoxide-sensitive DHE oxidation (Figure 2D). These results show that the unstable clones CS-9 and LS-12 appear to demonstrate increases in steady-state levels of H2O2 relative to the parental and 114 stable clones. Interestingly, the stable 118 clone showed what appeared to be some increase in H2O2 as well. However, since the intracellular redox status is also governed by antioxidant enzymes, the activities of primary antioxidant enzymes responsible for detoxification of superoxide (i.e. SOD) and hydrogen peroxide (i.e. GPx and catalase) were investigated.
Genomically unstable clones show increased steady-state levels of ROS
Antioxidant enzyme analysis showed that the activities of the H2O2-scavenging enzymes catalase and GPx were significantly elevated in the stable clones 114 and 118 relative to the wild-type and unstable clones (Figure 3A). On the other hand, catalase and GPx activities in the unstable clones were either reduced or comparable with the wild-type. Since GPx activity is affected by the intracellular availability of reduced glutathione, glutathione levels were evaluated in these cell lines (Figure 3B). Consistent with increases in GPx activity, the stable clones had increases in GSH levels relative to the wild-type. It had been proposed that the product of intracellular GSH and GPx activity gives a more accurate reflection of "effective GPx activity" [19], which is a better indicator of the H2O2-detoxifying capacity of the cell. On doing this calculation and propagating the errors in both measurements, we observed that the effective GPx activity was elevated in both of the stable clones (Figure 3C). In addition, Figure 3(D) shows that there was no significant difference in either MnSOD or CuZnSOD activities in these clones. The results of the antioxidant enzyme analysis suggest that hydrogen peroxide removal may play a more critical role (as compared with superoxide removal) in governing radiation-induced genomic instability.
Hydroperoxide-scavenging enzyme activity is increased in genomically stable clones
Together, these results suggest that increases in steady-state levels of H2O2 may be causally related to radiation-induced genomic instability. To test this hypothesis, the mutation frequency assay was repeated in the presence of 100 units/ml PEG-CAT using the unstable CS-9 cells. Figure 4(A) demonstrates a dramatic suppression of mutation frequency in the PEG-CAT-treated CS-9 cells, which also showed a 2-fold increase in catalase activity relative to untreated cells. When the experiment was repeated using PEG alone, no suppression of mutation frequency was observed (results not shown), providing clear evidence for the role of catalase enzyme activity in the suppression of the mutator phenotype. Furthermore, PEG-CAT treatment demonstrated a dose–response relationship with suppression of mutation frequency (Figure 4B). To confirm further the causal role of H2O2 in radiation-induced genomic instability, catalase activity was inhibited in the 114 stable clone using AT, and the 6-thioguanine mutation frequency assay was repeated. When 114 cells were grown in the presence of 20 mM AT for 4 days (85–90% inhibition of catalase activity) and then plated into 6-thioguanine for 2 weeks, mutation frequency was significantly increased 2–2.5-fold (Figure 4C).
H2O2 causes radiation-induced delayed mutagenesis
Since the mutator phenotype induced following exposure to radiation is believed to be a dynamic self-perpetuating process, the next series of experiments were designed to determine mutation rates (as opposed to mutation frequency) in populations of genomically stable and unstable clones with or without manipulation of catalase activity. The cell populations were cleansed of pre-existing mutations at the hprt locus by passage in complete media supplemented with 1×HAT for 3 weeks. Following HAT selection, cells were grown with or without manipulation of catalase activity for 10–15 days and then plated for the mutation frequency assay. Mutation frequency at a given time was divided by the number of days out of HAT selection to obtain the mutation rate. The results in Figure 4(D) show that the baseline mutation rate of the unstable clone (CS-9) was significantly higher than both wild-type (17-fold) and the stable clones (5- to 10-fold). Consistent with the hypothesis that H2O2 is causally related to the radiation-induced mutator phenotype, treatment with 100 units/ml PEG-CAT completely suppressed the increased mutation rate demonstrated by CS-9. In addition, treatment of the stable clones 114 and 118 with the catalase inhibitor AT significantly increased mutation rates to a level greater than the wild-type. In fact, the AT-treated 118 clone demonstrated mutation rates similar to the CS-9 unstable clone (Figure 4D). Interestingly, the 114 clone, which had lower steady-state levels of H2O2 (Figure 2B), demonstrated a smaller increase in mutation rate with AT treatment (Figure 4D) when compared with the stable clone 118, which had a higher baseline steady-state level of H2O2 (Figures 2B and 4D) and showed a greater increase in peroxide levels following AT treatment (results not shown). These results clearly show that radiation-induced genomic instability is a dynamic process that can be manipulated for many generations following radiation exposure by altering the activity of the specific H2O2 scavenging enzyme catalase.
Several theories have been proposed to explain the trans-generational nature of radiation-induced mutator phenotypes, with the "DNA repair hypothesis" being one of the most prominent [1,20]. According to this theory, a mutator phenotype can arise if exposure to radiation damages a critical gene coding for a protein that plays a role in DNA repair. In such a case, it is thought that cells would continue to show increased mutagenesis that would persist as a trans-generational phenotype. While some evidence in favour of this theory has accumulated [21], the small 'target size' presented by the genes encoding repair proteins, coupled with uncertainty surrounding the underlying source of spontaneous DNA damage, make it unlikely that repair genes are the only targets for radiation-induced mutator phenotypes (reviewed in [22]). Further support for this idea comes from past work showing the frequency of ionizing radiation-induced genomic instability in any surviving clone to be ∼3% per Gy [23], an incidence far too high to be explained by single (or even multiple) hit theories. It has also been shown by a rigorous microarray analysis that even though the genes associated with various signalling pathways are differentially down-regulated in the unstable clones, no single signalling pathway or repair gene correlated with a particular end-point of genomic instability [24].
Since genomic instability has also been associated with metabolic oxidative stress, a possible additional target for ionizing radiation-induced genomic instability could be genes coding for proteins involved in metabolic processes capable of producing ROS [3,6,22,25,26]. This hypothesis is supported by the fact that the progeny of irradiated cells show some evidence of dysfunctional mitochondria [6] and oxidative stress [4,6,7]. In addition, it was reported that pretreatment with free radical scavengers is capable of inhibiting the occurrence of radiation-induced genomic instability [27]. However, until now, studies identifying the specific ROS that cause persistent trans-generational radiation-induced mutator phenotypes were lacking.
The present study focused on identifying the role of O2•− and H2O2 in the mechanism of radiation-induced genomic instability. The results showed no consistent changes in the steady-state levels of O2•− or the superoxide-scavenging enzymes, CuZnSOD and MnSOD, in unstable or stable clones. In contrast, radiation-induced genomically unstable cell lines demonstrated significantly increased steady-state levels of H2O2. Analysis of genomically stable cell lines did, however, show a significant increase in the H2O2-scavenging enzymes GPx and catalase. When the unstable cells were treated with a cell-permeant specific scavenger of H2O2, PEG-CAT, mutation frequency and mutation rates were dramatically suppressed. In addition, when catalase activity was inhibited in stable clones, mutation frequency and mutation rates were significantly increased. These results conclusively identify H2O2 as the primary cause of the radiation-induced mutator phenotype in this model system.
The identification of intracellular H2O2 as being causally involved with radiation-induced trans-generational genomic instability provides a mechanistic backdrop for the mutator phenotype hypothesis and suggests the critical importance of oxidative metabolic processes [7,20]. The central dogma in mammalian cell biology is that genetic material (DNA) undergoes transcription and translation to make functional proteins that execute essential cellular processes (i.e. production of energy and reducing equivalents necessary for metabolism and biosynthesis). In Figure 5, we show a theoretical model by which oxidative metabolism leading to the production of H2O2 can be incorporated into the mutator phenotype hypothesis. In this model, radiation can damage genes coding for proteins involved in either oxidative metabolism or DNA repair. When radiation damages genes coding for proteins capable of resulting in increased levels of H2O2, a heritable change in steady-state levels of pro-oxidants leading to metabolic oxidative stress could occur. The resulting pro-oxidant intracellular environment could then perpetuate the process of mutagenesis, leading to genomic instability. Additional perturbation to the redox homoeostasis in cells could increase stress-induced damage to genes coding for DNA repair and further exacerbate genomic instability [20,21,28]. This model would predict a feed-forward loop where radiation-induced metabolic oxidative stress in concert with damage to DNA repair machinery could progressively accelerate genomic destabilization, leading to the plethora of deleterious biological effects associated with late radiation damage. This model also significantly increases the effective target size for heritable damage capable of leading to genomic instability, which is consistent with the prevalence and variety of unstable phenotypes that have been observed following radiation.
Incorporation of oxidative metabolism into the mutator phenotype hypothesis
The model in Figure 5 also predicts that cellular and dietary antioxidants specifically directed at mitigating the effects of hydroperoxides could rescue mammalian cells from this fate, effectively breaking the feed-forward loop and thereby slowing the progression of the radiation-induced mutator phenotype. Antioxidants can slow the rate of oxidative damage and might also allow for high-fidelity repair processes to work more effectively. The net result might be that, in the presence of antioxidants, high-fidelity repair capacity could offset the rate of damage, significantly reducing the progression of unstable phenotypes. Since genomic instability is thought to be an early step in tumorigenesis as well as degenerative diseases associated with aging, using specific scavengers of hydroperoxides to abrogate deleterious effects of radiation provides an attractive biochemical rationale for the development of strategies to mitigate a broad range of radiation-induced effects.
This work is supported by the U.S. Department of Energy and NIH grants DE-FG02-05ER64050, RO1CA100045 and P30-CA086862. We thank Ling Li, Mitchell C. Coleman and Nükhet-Aykin Burns for discussions regarding the antioxidant enzyme assays, Kjerstin M. Owens for discussions related to the mutation rate assay and Melissa A. Fath for discussions regarding the EPR protocol and Ehab H. Sarsour for editorial assistance. We also thank the Flow Cytometry facility and the ESR facility at the University of Iowa.
carbamoyl-P synthetase/aspartate transcarbamylase/dihydro-orotase
- c-DCF
5-(and 6-)carboxy-2′-7′-dichlorofluorescein
- c-DCFH2
5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate
5,5-dimethyl-1-pyrroline N-oxide
- GPx
glutathione peroxidase
Hanks balanced saline solution
- hprt
hypoxanthine–guanine phosphoribosyltransferase
Nitro Blue Tetrazolium
polyethylene glycol
PEG-conjugated catalase
- p-HPA
p-hydroxyphenylacetic acid
reactive oxygen species
superoxide dismutase


text: The festive season of Christmas is fast approaching and all of the adverts on TV are already Christmas themed. CMA Country performed a Christmas special and all the fans loved it. All of the biggest country stars took to the stage to deliver a incredible performance. Reba McEntire and her four members of Little Big Town, Karen Fairchild, Jimi Westbrook, Kimberly Schlapman and Phillip Sweet take to the stage and start singing the words of "Mary Did You Know".


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Our steel provides propulsion
Mobility is a basic human need, and one of the main driving forces behind technological development. There are many companies that help people and goods get from A to B – by land, water or air. By supplying these companies with its steels and products, GMH Gruppe operates as an innovative partner: fast, flexible and always looking for new solutions and fields of application.
The automotive industry – we keep everything running smoothly
Steel is the fuel that drives the automotive industry. And GMH Gruppe is one of this industry's leading suppliers. Around 80 per cent of our steel is used by the international automotive industry and its suppliers. Our steel is used to make crankshafts, cam shafts and drive shafts, as well as gearbox and chassis parts for cars and lorries. Our premium-quality cast iron serves as the basis for engine parts, exhaust manifolds and transmission components.
Lightweight construction – efficiency driven by steel
GMH Gruppe is also actively involved in the field of lightweight construction. This issue is becoming increasingly important to future development, especially in the automotive industry. By 2030, the percentage of high-tensile steels in cars will rise from 13 to 38 per cent. This is because it is the most important material for economically viable lightweight construction. The overall weight of vehicles can be significantly decreased by reducing the weight of several different components. This contributes significantly to reducing CO2 emissions.
In order to research the potential of lightweight construction for the car industry in more detail, we joined the cross-industry Lightweight Forging Initiative in 2013. This initiative has developed several new ideas for reducing the weight of cars and commercial vehicles. It has also demonstrated that solid lightweight construction solutions using high-performance steel are both cost-effective and competitive. With more than 500 ideas for over 2,500 parts, lightweight components are already hugely important to the automotive industry – and will play an even greater role in the future.
Shipbuilding – sailing straight and true
Customers in the shipbuilding industry also rely on GMH Gruppe products. We produce a number of open-die forged parts for this sector – from raw steel bars and ready-to-install crankshafts to large crankshafts and cast components for large machines.
Rail transport operations – quality that keeps you on track
GMH Gruppe has close ties with the rail sector. The modern, sophisticated rail transport sector requires manufacturers to continuously optimise their product development and manufacturing processes. As such, our main goal is to fulfil all the relevant safety and quality requirements – thus ensuring a high level of customer satisfaction. To guarantee that we achieve this goal. we deploy the latest manufacturing methods and simulation technology, and exclusively use materials that have beensubjected to stringent quality control. Our products, consequently, stand for consistent quality, high availability, economic efficiency and excellent longevity.


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In response to needs for in situ thermometry, a temperature-sensitive vector was adapted to report changes in the intracellular heat content of Escherichia coli in near-real time. This model system utilized vectors expressing increasing quantities of β-galactosidase in response to stepwise temperature increases through a biologically relevant range (22 to 45°C). As judged by calibrated fluorometric and colorimetric reporters, both whole E. coli cells and lysates expressed significant repeatable changes in β-galactosidase activity that were sensitive to temperature changes of less than 1°C (35 to 45°C). This model system suggests that changes in cellular heat content can be detected independently of the medium in which cells are maintained, a feature of particular importance where the medium is heterogeneous or nonaqueous, or otherwise has a low heat transfer capacity. We report here that the intracellular temperature can be reliably obtained in near-real time using reliable fluorescent reporting systems from cellular scales, with a 20°C range of detection and at least 0.7°C sensitivity between 35 and 45°C.
Temperature is a master environmental variable, whose accurate measurement is critical for understanding biological processes from the molecular to the biome level (13, 15). At a molecular level, temperature in biological systems is typically measured by observing the surrounding growth medium, and while the rapid thermal relaxation of the cellular milieu and aqueous medium makes this approximation valid under many circumstances, there are notable exceptions. Rapid cellular and subcellular heat content changes have been theorized to result from alternating electromagnetic fields (EMF) (7), and the continued development of hyperthermia-based therapies combining nanomaterials and EMF (11) requires the development of in situ and in vivo temperature measurement for subcellular and tissue levels.
Cellular heat content affects biomolecular assembly, structure, and function. It has long been recognized that cellular systems sense temperature through various means, including transcription factor binding and mRNA secondary structure (3, 5, 13, 15, 16, 21). In vivo, these systems maintain homeostasis by responding to environmental temperature changes (5, 6, 16, 20–22). Subtle changes in biomolecular structure alter the temperature response, as evidenced by temperature-sensitive mutations (2). While these systems have been engineered or are otherwise exploited for functional studies and expression control, none of these heat-sensing systems have been developed for intracellular thermometry (15). We report here the first demonstration of a biomolecular intracellular thermometer. This system utilizes temperature-sensitive mutants of lacI controlling LacZ expression (3, 4), which accurately reports cellular temperature in an environmentally relevant range.
Constitutive expression of LacI(Ts) by a T7 promoter (derived from pET24; Novagen/EMD, Darmstadt, Germany) results in the availability of LacI(Ts) to bind lacO sites. At permissive temperatures, where LacI(Ts) is bound to the two lacO sites engineered into a T7A1 promoter, transcription of lacZ is blocked (Fig. 1). As the intracellular heat content is increased, structural changes cause LacI(Ts) to leave the lacO sites, leading to LacZ expression (Fig. 1). LacZ (β-galactosidase) then cleaves the lactose analog 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal; Mallinckrodt Chemicals, Phillipsburg, NJ) into subunits that then react with one another to form an insoluble blue precipitate (5,5′-dibromo-4,4′-dichloro-indigo), a colorimetric indicator of LacZ activity. In addition to the conventional colorimetric assays, 5-dodecanoylaminofluorescein di-β-d-galactopyranoside (C12FDG; Invitrogen, Eugene, OR) can be utilized as a fluorogenic substrate for LacZ. C12FDG has 2 galactoside residues that are sequentially cleaved, yielding a fluorescein moiety that is retained in the cell membrane by its hydrophobic tail (23). These LacZ reporters were successfully employed to assay the intracellular temperature response.
The biomolecular nature of this temperature-sensing system reflects a fundamental manner in which other cellular components are likely to respond to intracellular heat content changes. When related to a relevant scale, in this case degrees Celsius, this heat-sensitive expression system becomes a viable thermometer. We demonstrate a colorimetric biomolecular thermometer with a physiologically relevant functional range between 25 and 45°C and with a sensitivity better than 0.7°C (35 to 45°C). LacZ cleavage of X-gal can be detected in less than 30 s after exposure to 40°C. Further, the fluorogenic reporter and LacZ substrate C12FDG can be used to identify individual cells that have been heated by fluorescence microscopy and can be readily applied to fluorescence-activated cell sorting (FACS) (12) and possibly even whole-body imaging (14).
Cultivation. (i) Strains and culture.Escherichia coli DH5α strains harboring the LacI(Ts) vector pA199A2-Its187 (Its187), pA199A2-Its233 (Its233), pA199A2-Its241 (Its241), or pA199A2-Its265 (Its265), carrying a Gly187Ser, Leu233Lys, Ala241Thr, or Gly265Asp point mutation in the lacI gene, respectively (3, 4), were isolated as individual colonies onto Luria-Bertani (Miller formulation) agar (LB agar; EMD Chemicals, Gibbstown, NJ) with 100 μg/ml carbenicillin (MBI Growcells, Irvine, CA). These plates were cultured at room temperature (RT; 22°C ± 1°C) for 48 h. A liquid culture of each strain was inoculated from a single colony. Five milliliters of each culture was incubated at RT with shaking at 135 rpm in LB medium with 100 μg/ml carbenicillin for 48 h. One milliliter of this initial liquid culture was then transferred to 200 ml LB medium with 100 μg/ml carbenicillin and was grown for 48 h with shaking at RT.
(ii) Chemostat.For each of the four E. coli strains, a 2-liter chemostat, with a working volume of 150 ml of LB medium with 100 μg/ml carbenicillin and D of 1.3 h−1 (flow rate, 200 ml/h), and with shaking at 135 rpm at RT, was inoculated with 50 ml of the culture described above and was allowed to equilibrate for at least 16 h.
Assays.Cultures were washed twice with phosphate-buffered saline (PBS). They were then normalized by spectrophotometry to an optical density at 600 nm (OD600) of 0.05 for the initial characterization and by direct microscopic count (10, 18) to 5.5 × 109 cells/ml for all subsequent experiments utilizing the colorimetric assay. Five microliters of 8 mg/ml X-gal in dimethyl sulfoxide (DMSO; Fisher Scientific, Fair Lawn, NJ), freshly prepared, was added to 50-μl aliquots of cells, which were immediately placed in a MiniOpticon PCR machine (Bio-Rad, Hercules, CA) for heating at controlled temperatures. For the initial examination of thermal sensitivity, the block was held at discrete temperatures (±0.2°C) sequentially increased in 5°C steps in the range from 25 to 45°C, in addition to an RT control (Fig. 2). Once the mutant with the largest response was determined (Its265), the gradient function, with upper and lower limits of 45 and 35°C, respectively, was utilized (Fig. 3). Cell suspensions with X-gal were heated for 20 min, and then the cells were lysed and the reactions were stopped by the addition of 50 μl 1 M sodium carbonate (Sigma-Aldrich, St. Louis, MO). To observe the characteristic kinetic window, a cell suspension of Its265 with X-gal (as described above) was heated in a 40°C water bath (Precision Scientific Inc., Chicago, IL) for 30 s to 20 min and was then transferred to the PCR machine block at 35°C for an additional 20 min before the reaction was stopped. To confirm that the colorimetric responses observed were significantly greater than what could be associated with increases in LacZ/X-gal kinetics due to the Arrhenius effect, a subset of reaction mixtures were heated prior to the addition of a cell-disrupting agent, toluene, to fix the LacZ levels, after which X-gal was added and all reaction mixtures were held at the same temperature.
All reaction mixtures were then incubated at 4°C for at least 12 h before colorimetric analysis to allow completion of the reaction of cleaved X-gal with the insoluble blue precipitate. This insoluble blue precipitate was quantitated spectrophotometrically with 2 μl of the well-mixed reaction mixture by measuring the OD630 on an ND-1000 spectrophotometer (NanoDrop, Wilmington, DE). Two microliters of a mixture of 50 μl PBS, 5 μl DMSO, and 50 μl sodium carbonate was used as the blank for spectrophotometry.
For the fluorometric assay, cells from the chemostat were diluted 10-fold into LB medium containing a final concentration of 20 μM C12FDG and were incubated for 1 h at RT with shaking at 135 rpm (procedure modified from flow cytometry as described in references 12, 17, and 23). An aliquot of cells was heated in a 40°C water bath for 20 min, while an aliquot of control cells was held at RT for 20 min. Cells were then filter immobilized on 0.4-μm-pore-size, 25-mm-diameter black polycarbonate membranes (GE Water & Process Technologies, Trevose, PA), counterstained, and mounted with 0.2 mg/ml 4′,6-diamidino-2-phenylindole, dilactate (DAPI; Invitrogen, Carlsbad, CA), in 95% glycerol. E. coli strains were then photographed/observed using a Nikon Eclipse E400 series epifluorescence microscope (Nikon Corp., Tokyo, Japan). Images were captured by a Spot Insight QE tricolor charge-coupled device (CCD) camera (Diagnostic Instruments Inc., Sterling Heights, MI) connected to a personal computer (Dell, Nashville, TN). Image analysis was performed using ImageJ (NIH, Bethesda, MD).
Statistical analysis.Statistical analyses were performed using Microsoft Excel 2004 for Macintosh (version 11.5.5; Microsoft Corp., Redmond, WA). Averages presented represent the arithmetic mean. Standard deviations presented were calculated using the n − 1 degrees of freedom.
Comparison of the four LacI(Ts) strains.Mutagenesis screens for the LacI(Ts) strains described above and other LacI(Ts) mutants are typically conducted with a 30°C to 40 or 42°C shift on X-gal-containing plates; colonies that are white at 30°C and blue at 40 or 42°C are temperature-sensitive mutants, and white/white or blue/blue colonies are wild type or have loss-of-function lacI mutations, respectively (1). In order to characterize these temperature-sensitive vectors in the context of a thermometric assay, a broad yet physiologically relevant temperature range between 22°C and 45°C was tested. For E. coli, this represents the range from the lowest practical culture temperature (RT) to the temperature at which the heat reservoir begins to affect viability (45°C) (19). All four strains carrying the LacI(Ts) vectors Its187, Its233, Its241, and Its265 were cultured, washed, mixed with X-gal and DMSO, and heated for 20 min at 5°C intervals in independent triplicates.
In all cases, the cultures responded to increasing temperature with increased expression of LacZ, as colorimetrically indicated by the insoluble blue product (Fig. 2, inset). As expected, each of the strains responded in a unique manner to the temperature increase; Its187 and Its265 were far more sensitive in this temperature range than Its233 and Its241 (Fig. 2). Its265 was thus utilized for all future experiments, because of its sensitivity and lower baseline expression of LacZ.
Its265 sensitivity.A narrowed temperature range of 35 to 45°C was examined in order to determine reporting sensitivity using E. coli carrying pA199A2-Its265. The test was carried out utilizing the PCR gradient function of the MiniOpticon, with the temperatures in each row of the block corresponding to 35, 35.7, 37.1, 38.9, 41.2, 43, 44.3, and 45°C according to the accompanying gradient calculator software (Bio-Rad, Hercules, CA). The test was carried out three times with five independent replicates (n = 15) (Fig. 3). All responses were normally distributed, and significant differences (P ≤ 0.05) between colorimetric distributions at each temperature, observed using a two-tailed paired Student t test, indicate an assay sensitivity better than 0.7°C. In accordance with the observations of Chao et al. (3), the increases in LacZ production and activity, and in associated X-gal metabolism, were significantly greater than what could be attributed to increased kinetics in accordance with the Arrhenius effect. This was confirmed experimentally by heating and then disrupting the cells with toluene prior to the addition of X-gal, after which all reaction mixtures were held at the same temperature. The cell lysates demonstrated a significant increase in OD630 with heating (data not shown).
Time course of LacZ induction.A time course experiment was carried out to determine how quickly a significant increase in LacZ expression could be detected. To measure LacZ expression, E. coli Its265 was incubated at 40°C in 4% DMSO with X-gal for 0 and 30 s and 1, 2, 5, 10, 15, and 20 min (repeated in at least triplicate). A subsequent incubation for 20 min at 35°C permitted any LacZ expressed during the 40°C exposure to catalyze the cleavage of X-gal and resultant formation of the insoluble blue precipitate. Spectrophotometric detection at 630 nm indicated a significant difference after 30 s (P = 0.013). Little difference was seen between 30 s and 2 min, followed by a steady increase in LacZ expression over the next 18 min (Fig. 4).
Fluorometric reporting.Several indicator substrates are available for LacZ, including a wide variety of colorigenic and fluorogenic substrates. These are available with various chemical modifications that necessitate different methods of cellular introduction (loading). The particular fluorogenic substrate utilized in these studies, C12FDG, is a fluorescein molecule with 2 galactose molecules attached by β-galactoside linkages that can be cleaved by LacZ, leading to a fluorescent product, and a 12-carbon lipid tail that partitions into the cell membrane and serves to hold the cleaved product (23).
Following a 1-h incubation with C12FDG at RT to load the cells with the substrate, the cells were aliquoted into a heated (40°C for 20 min) fraction and an RT fraction. Cells were then filter immobilized for fluorescence microscopy and were mounted in 95% glycerol containing 0.2 mg/ml DAPI. Photomicrographs were taken using the same exposure settings for heated (Fig. 5B) and nonheated (Fig. 5A) samples. A mixed sample, containing a mixture of equal parts of the RT control (minimal fluorescence, yellow arrows) and heated cells (increased fluorescence, red arrows) was captured using different exposure settings in order to successfully juxtapose a broad range of intensities (Fig. 5C). Contrast adjustments were made using ImageJ (solely to reduce background), and all optical software adjustments were identical for all images captured. Differences in the fluorescence intensity are readily visible in response to the temperature exposure history, both in the separate micrographs and when RT and heated cells are mixed together in equal parts (Fig. 5), demonstrating the viability of this thermometric method where a fluorescence-based methodology would be beneficial.
Prior to this study, the general approach to gauging intracellular heat content relied on measuring the temperature of the surrounding medium, assuming that the rapid thermal relaxation of a cell-medium system was such that the temperature cells experience would be uniform. As contextual challenges to this practice evolve (7) and therapies relying on accurate application of local heating have developed (11), the need for molecular-level thermometry in biological systems is an emerging and important frontier (15).
Recently, molecular-level thermometry has been demonstrated using fluorescent nanohydrogels (8, 9). While nanogels are effective for observing temperature changes at an intracellular level, their effective range and sensitivity are limited, and mid- to long-term biocompatibility studies have yet to be conducted (8, 9). We report here an alternative approach to intracellular thermometry, with a 20°C range of detection and at least 0.7°C sensitivity between 35 and 45°C. Furthermore, this method relies on temperature-sensitive mutants of native proteins, eliminating concerns about biocompatibility with the sensing mechanism. With a vast array of LacZ substrates—some of which have been utilized for decades—biocompatibility for reporting can be tailored to a variety of applications.
Adaptation of these vectors to other cellular systems is envisioned, since expression systems using the Lac operon have been developed for virtually every model system from bacteria to humans (17). Further, alternate genes can be engineered in place of the lacZ gene to provide different indicators; enhanced green fluorescent protein (EGFP) could be inserted, yielding a fluorescent reporter with low toxicity and no required indicator substrates. Development and characterization of this construct are under way.
One drawback of a biomolecular thermometer, and especially multistep expression-based systems, is the variability in the growth rate, cell stage, and metabolic state, which may affect the quantitative outcome of this type of assay; thus, standards under experimental conditions must accompany each experiment so as to provide a calibrated result. Again, it should be noted that this "drawback" results from the fact that the biomolecular thermometry better senses the effects of heating on the cellular milieu. The responses of cells to temperature changes will likely differ according to these culture conditions, permitting this method to better reflect the overall cellular response to heating. To a large extent, calibration variability can be mitigated with carefully controlled cultivation conditions, employing modern chemostats.
Conclusions.Temperature is a master environmental variable that affects all biological activity. Conventional temperature monitoring relies on averaged material responses to heat that we detect visually or with the aid of simple electronics. Because thermal conduction is relatively rapid in most aqueous systems, we typically observe the temperature of the medium (or other environment) that hosts the cells of concern—and accept this as a relatively accurate surrogate for the temperature within the cells themselves. There are, however, a multitude of situations where temperature measurements of the immediate environment are impractical. Further, intercellular temperature may differ from the near-field surroundings on significant scales, particularly where large density differences exist between microorganisms and their environmental interface. Solid (polymeric) surfaces, filter media, and membranes constitute germane examples of common bioengineering applications where the presence of microbes in or on an environmental feature does not necessarily result in shared heat content in important time frames.
With respect to in situ biochemical conditions, procedures enabling cells to report nondestructively on certain aspects of their immediate status have been employed for decades. Indeed, vectors have been leveraged to report on a wide variety of intra- and intercellular metabolic and signaling activities; only relatively recently, however, have they been recruited for physical factors, including, but not limited to, microscale temperature assessment. If intracellular genetics-based reporters with adequate reporting sensitivity are engineered with optically active elements that do not require chemical primers, a cellular-scale level of physical temperature measurement may soon be available in real time.
This work was supported by NIH grant RO1 EB 009115-01 A1, the State of Colorado, and the Leadership Alliance.
- Received 11 August 2010.
- Accepted 16 February 2011.
- Accepted manuscript posted online 4 March 2011.
- Copyright © 2011, American Society for Microbiology


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REEF, with the ongoing support of local businesses, is continuing its monthly lionfish contest that awards both groups (shops, clubs, teams) and individuals who turn in the most lionfish each month. This is a fun competition to encourage ongoing removals of invasive lionfish.
The seasonal fishing ban on endangered Nassau grouper spawning aggregation sites in the Cayman Islands, which was set to expire at the end of this month, has been extended for another eight years.
Divers removed 312 invasive lionfish from Florida Keys waters during the final 2011 Florida Keys Lionfish Derby event last Saturday, Nov.
Reef Environmental Education Foundation (REEF) in partnership with Miami-Dade County Sea Grant Extension will be organizing a series of lionfish workshops open to the public for the Southeast Florida area.
The Reef Environmental Education Foundation (REEF), headquartered in Key Largo, has appointed Martha Klitzkie as general manager. Klitzkie began her career as education director at the Pigeon Key Foundation in Marathon. She went on to direct a l
Divers successfully removed 675 invasive lionfish from Florida Keys waters on August 20, during the upper Florida Keys derby of the Second Annual Florida Keys Lionfish Derby Series in Key Largo, Fla., organized by Reef Environmental Education Foundation (REEF) and the FL Keys National Marin
A good time was had by all who participated in the Third Annual Lionfish Derby at Green Turtle Cay, Abaco, Bahamas on June 24th and 25th, 2011. The event was held at the Green Turtle Club, whose sponsorship, along with the Bahamas Mini
The Reef Environmental Education Foundation (REEF) will launch the 19th Great Annual Fish Count (GAFC) with an open house and presentation at its headquarters in Key Largo, Florida Tuesday July 12th at 6:30 PM.
We are honored to release the 2010 REEF Annual Report. The report is posted online at www.REEF.org/reef_files/annual2010.pdf.


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Giovanni Bove is Senior Editor of Technology. In addition over the past several years Gio has served as an IT specialist for the Eye of the Hurricane. Gio is a member of the National Honor Society and has been very involved in the STEAM program.
Gio Bove, Senior Editor-Technology
|Jun 06, 2019
Mrs. Hogsett The Best a Teacher Can Get!! FALL PREVIEW 2019 (Story)
|May 17, 2019
Maria Owens Triple Jump Champion! (Story)
|May 23, 2018
Getting to Know Mrs. Vournous with Just 9 Questions (Story)


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If the dark cloud of divorce is looming over your Valentine's Day, you're not alone — many single parents struggle with it. It might seem like the love-themed holiday isn't for you now that you're on your own, but that's not true. You have a lot of love in your life from your children, and that is certainly something worth celebrating.
Why Choosing to Co-Parent Makes You A Better Mom
I've heard it over and over again from my single mother friends, "...joint custody totally sucks. I hate not seeing my kids." I absolutely sympathize with mothers who loathe missing out on 50% of their child's life. That's A LOT of time to be away from your trusty little sidekick(s). And I can't say that I can empathize with you, because I actually have no idea what it feels like to split custody with another parent. However, I do understand what it feels like to be on the other side of the spectrum.
20 Tips for Single Parents Traveling with Young Children
Any parent knows that traveling with young children is...challenging. At times, it can be downright frustrating, especially if you're a single parent. But if there's one thing parents are known for, it's how to be resourceful. Here are some tips to make globe-trotting with children easier and a heck of a lot more fun!
Bottles & Brains: Pursuing Education as a Single Parent
I was only one semester into graduate school when I found out I was pregnant with my first son. Was I scared? Was I worried about how I would afford daycare and diapers? Did I have doubts about how to juggle an education, a career and a family? Of course! I felt completely overwhelmed, but I never once considered giving up.
6 Things You Should Never Ask an Unwed Mom-To-Be
Love, Marriage, Baby. It's a beautiful trio, but the order in which each item appears is not always so clear. Whenever there is a pregnancy and the couple isn't married, there are a hundred burning questions that people want to ask, but feel like they shouldn't. So most of the time, people either make up stories or assume things that may (or may not) be true. Here is what NOT to ask.


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Letter 85 is the eighty-fifth letter written by J.R.R. Tolkien and published in The Letters of J.R.R. Tolkien.
Summary Edit
Tolkien reported battling with the separated sequence of the Ring, which had meddled with other dire and more blunt obligations and had ceased his composition progress. His answers were little guide changes, an additional day for the Entmoot, and additional days for Trotter's (Early name of Aragorn) pursuit and Frodo's voyage (two days from the Morannon to Ithilien). Shockingly he needed to stop and convey addresses.
Community content is available under CC-BY-SA unless otherwise noted.


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February 1, 2019
It's been a very busy time here at PlayFusion, and we are thrilled at the response to our PC and Switch releases.
Despite fantastic feedback to our Switch launch, we understand a major priority has been for us to develop and release controller support for the Switch. Because of this we are looking to implement Classic mode during next season and thank you all for your patience.
We wish to implement classic mode alongside our Switch controller update – So all our players can enjoy the best and latest experience at the same time.
Changes regarding Classic
With Classic's release, this will replace any existing formats ( across all game modes )
If you're new to Lightseekers or want to know more about Classic Mode you can read more here
After Uprising's release, all formats will remain Classic bar ranked; this will be replaced with the Standard format – which simply put is to build decks from the cards that are not retired or banned. You can read more about this in our tournament policy.
As always we really appreciate your continued feedback and can't wait to see the community grow this year with our planned releases.


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Homeschool family or not, if you discuss homeschooling, the "S" word eventually pops up.
S o c i a l i z a t i o n
*insert eye roll here*
Answering the socialization question has become routine for me. The usual answer, "Have you met my children?" I have extremely social children and have no doubt that they will grow up to be properly socialized adults. My hope is that they will see people through the eyes of Jesus – something you can't necessarily teach in a classroom. Enough about the children, however.
Mr. Sweet and I recently went out of town for the weekend for a special event without the kids. He works very hard to support our family and it was nice to have a break from the normal routine. At one point I found myself talking incessantly and it hit me. Socialization isn't just for the kids. The homeschool parent needs it, too!
"The activity of mixing socially with others."
Stay at home moms and dads, when was the last time you mixed socially with others?
My thoughts, exactly.
We spend countless hours enriching our children (sometimes just helping them survive) and forget that we need a little interaction, too.
Be intentional about your adult socialization. Go on a date with your spouse. Talk to each other. Plan a coffee hour with other homeschool moms (without the kids). Talk to the homeschool moms who are waiting on kids at art class, instead of sitting in your car. Ok, that was a personal example 😬 but you get the point.
Socialization…not just for the students.


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1900 Village West Pkwy
Kansas City, KS 66111
The Kiwanis Club of Kansas City, Kansas will have their FREE Bike Rodeo event on Saturday, May 18 between 9AM and noon. Held at T-Bones Park, this day is all about bike safety, as families can participate in bike safety courses and activities. The first 300 kids will receive FREE bike helmets and t-shirts. This is not just for Wyandotte County as any elementary aged child can come and bring their bike!
There are also sponsorship opportunities available for businesses. See below.
Joe Vaught is an active/charter member of our club, and helps us annually with this rodeo, so he can also answer questions!
Here's the flyer for the event. Feel free to share it at your business.
Bike Rodeo 2019
There are also sponsorship opportunities. Sponsorship form is below.
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What are your strangest writing habits? Ones that you find yourself doing either because they help you personally with the writing process or just because of your own strange personality
Drinking lots of coffee while listening to electro house music. Sometimes I even drink energy boosters with coffee too.
I listen to Old School Hip Hop, Hindu chanting, playlists (GTA, Jurrasic Park, Tetris Remix compilation, Panic Room, ect).
My dreams are also extremely helpful, I have dreams that are so crazy I doubt my own sanity sometimes.
Also, if I don't have Aztec or African art next to me, I might not be able to write at all.
I often want until its dark and then lock myself in my room and listen to noise music and shoegaze to get me into the proper writing atmosphere. That's how I wrote my most successful article, so I've just started doing that for everything.
Probably extremely tame, but even when I'm writing via keyboard, I still need a pencil or a pen or something to twirl around in my hand while I'm thinking.
Only nerds have signatures
Thank god I am not the only one that does that.
I can't be in my house when I want to write something. I'm always writing at work or someone else's place if I'm staying the night and everyone's asleep.
I write best without any organization. It's a pretty bad habit, but I HAAAAAATE having to plan out everything. It just… Feels wrong. I make up everything as I go, and I don't like having to see into the future.
Another thing: I prefer the good ol' pencil and paper over a computer. Maybe it's because I just can't sit still and.. Type. My mind wanders around, and I doodle a lot.
Personally I prefer to type as I'm the kind of person who's ideas will disappear quickly if I don't write them down. Because of this I write down most of my ideas on my phone rather than a notepad.
I have a hard time writing if my body is at rest. If I sit to write, I swivel the chair excessively. I prefer to stand up to write, though I know that's not great on my feet combined with all the standing I do at work, and I pace around a lot in-between paragraphs or sentences. I don't like silence during most anything, so I usually listen to retrowave or vaporwave playlists on YouTube while I'm at it.
I also have trouble coming up with a narrative to begin with if I don't take a run and think up the movie scenes in my head while I'm listening to whatever fitting music and getting in the zone.
Edit: Also can't focus at all unless I'm typing. I can't handwrite fast enough to remember sentences as my brain makes them up. I will handwrite outlines and bullet points for drafting, though.
It's nice to see that I'm not the only one who fidgets a whole lot while they're writing. If I write while sitting, I start with both feet on the floor and end with both legs numb because they've been wedged under my ass for the better part of an hour.
Where it won't be awkward, I've started talking to my phone Dale Cooper-style to try and work out kinks in whatever I'm writing. I don't actually bother listening back to the recordings; the idea is solving problems through describing them, a la rubber duck debugging. It's also a hell of a lot faster than handwriting, especially now that it's winter and my fingers cramp after a whole line of scribble.
My excoriation disorder gets really bad while writing, for whatever reason, so my fingers usually end up all sore and gross from me anxiety-chewing them. You probably never wanted to know that, but you do now.
I do the dishes while I'm brainstorming tale ideas.
This is more indicative of my general inability to sit still, but I rock back and forth in my chair whenever I'm writing. It's very much a subconscious act, but if I'm ever sitting still I can't write because I'm putting all my focus on not rocking.
… I pace around a lot in-between paragraphs or sentences.
Me all over. I also pace like mad or take laps around the building while trying to get everything in order in my head before even writing the first line, which is not a great habit.
I also spin my chair, roll back and forth from my desk, and fidget a lot in general.
This so much. I need constant activity or else I'll get bored. I need breaks in between writing sessions in order to let my brain foster the ideas I've already written down and see which ones do and don't work. If I don't I'm sure my writing will suffer because, out of all my other articles, the ones where I have done this are my most successful.
Although, I don't really have a specific genre I listen to while writing. Anything that has a catchy beat or just generally sounds nice to me I'll play it.
This is fundamentally how I write, too. Standing up, pacing, music
Before I do any of that, I walk around in circles hypnotizing myself by twirling a big tasseled scarf until a name, phrase, or title pop into my head.
I write the way Collin Firth talks in The King's Speech— sometimes when I can't think of a line, I'll write curse words in the place of the line until something comes to me.
I usually try to write stoned.
I usually have a cup of fruit juice or tea nearby.
*gasps* The humanity!
/me siiiiiiiip.
I don't finish what I started. To this date I've only managed to finish one out of twenty or so drafts.


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Request A Consultation
Request A Consultation
Drinking & Biking – The Law and DangersJuly 30, 2018| By: Jordan Kofman
If you decide to drink, do not bike. However, if you choose to ignore this advice, it is important...
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Beautiful ocean-view custom-built impeccably maintained home on pool-size lot! Elegant & charming w/ amazing curb appeal, exterior stone & artisan lighting. Main floor has formal living and dining rooms, kitchen w/ eating area, family room w/ fireplace, den/office (4th bedroom) & bath w/ walk-in shower. 2nd story has master suite, 2nd bedroom w/ full bath, 3rd bedroom w/ ocean view, hall bath w/ walk-in shower, bonus/media/game room w/ fireplace (5th bedroom), & laundry room w/ utility sink & built-in cabinetry. Gourmet kitchen has granite counter tops, travertine back splash, under-cabinet lighting, walk-in pantry, huge island, SS Viking appliances. Master suite has ceiling fan, fireplace, walk-in closet, bath w/ double sinks, separate tub & dual-headed shower w/ glass enclosure. French doors lead from living room & from master suite to front outside porches to enjoy the ocean breeze, & from family room to entertainer's back patio to enjoy the amazing mature landscape. Travertine floor downstairs & in all the baths & laundry room, plush neutral color carpet on stairs, upstairs hall & all bedrooms. Exquisite details include 10' trey ceilings w/ amazing crown molding, 8' Mahogany doors,7.5" baseboards, cathedral ceilings, ceiling fans, recessed lighting, Roman blinds & designer's paint throughout. Carriage porch, 2-car garage w/ attic space, huge backyard & lighted/gated dog run. Beach location close to shopping & entertainment. No HOA dues & no Mello-Roos. This is a must see!
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Based on information from California Regional Multiple Listing Service, Inc. as of October 18th, 2019 at 12:17am PDT. This information is for your personal, non-commercial use and may not be used for any purpose other than to identify prospective properties you may be interested in purchasing. Display of MLS data is usually deemed reliable but is NOT guaranteed accurate by the MLS. Buyers are responsible for verifying the accuracy of all information and should investigate the data themselves or retain appropriate professionals. Information from sources other than the Listing Agent may have been included in the MLS data. Unless otherwise specified in writing, Broker/Agent has not and will not verify any information obtained from other sources. The Broker/Agent providing the information contained herein may or may not have been the Listing and/or Selling Agent.


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If you would like more information please email Roger Kaye at
Quick Download Links
The Crusaders of Cumberland Court
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The Teenagers' Nativity
Free musicals - why?
The purpose of this site is to make five free musicals available:
The Crusaders of Cumberland Court
"Mamma Don't Marry That Man" Free Musical - Script Samples
ACT 1 - Scene 1
Mr Fisher - (formally) Now children, just before we go today I have a little brown envelope for each of you. You all know what's inside don't you?
Class - Yes Mr Fisher.
Mr Fisher - I've written your names on the top left hand corner of your envelope but I want you to write your parents' address on the envelope, just in case anyone happened to drop it on the way home. And we don't want that to happen do we Twinkle?
Twinkle - No Mr Fisher. (the class snigger)
Mr Fisher - I am afraid I have to rush off now to St Margaret's for an important sports fixture meeting, so when you've addressed your envelopes you can go.
Mr Fisher gives each person an envelope as he walks from stage left to stage right.
Remember that the envelopes must be addressed to your parents and they must be the first to open them.
Class - Yes Mr Fisher.
As soon as Mr Fisher exits stage right the students leave their places rush to the centre of the stage. Charlotte waves a fresh set of envelopes in front of them and they immediately tear open their envelopes and throw them in the air after first extracting their reports. Charlotte gives everyone a fresh envelope as they read what Mr Fisher has written about them and start to compare reports.
Ryan - (punching the air and giving a loud shout) Ohyes! Dis man is on my team. Check this – (reading from his report) 'Ryan has made sound progress in all subjects but has excelled on the football field, proving to be one of the most promising players I have coached in years'. Dat guys, is gonna get me a new skateboard. Bring it on.
Tyler - Fish Face says that I have one safe pair of hands, cos the other teams only put 5 goals past me all season.
The two boys give each other a high five. They then turn their attention to Twinkle who slumps to the floor with her head in her hands
Ryan - Oh no, what have we here? Twinkle is not twinkling today. The chicklet is not chuckling. Not such a good one eh Twinkle?
Charlotte - (putting her arm around Twinkle) Leave it out Ryan. Can't you see she's upset. Twinkle, whatever is the matter?
As Charlotte pulls Twinkle to her feet the instrumental introduction of the song plays. The students then form up around her and begin a dance routine as she sings to Charlotte.


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Gather round children. Do we have a story for you.
It's about a questionable, but reportedly very common method for relieving a baby's chest congestion that's gone viral because, well, you'll see.
Earlier this week, American "breastfeeding and pregnancy support and education" company Melons and Cuties shared a photo of a parenting hack from one of their customers on Facebook.
"Our customer shares her remedy for chest congestion!! Use on baby's back while sitting in a steamy bathroom. Brilliant!!!" the caption read alongside an image of a newborn baby in an adorable leopard print onesie, with a vibrator held against its back.
Yes, the same kind of vibrator you may or may not be intimately familiar with.
Since it went live on Tuesday, the viral post has over 10,000 comments, a large majority of which are supportive, hilarious and deeply traumatising.
"HILARIOUS!!! But also a BRILLIANT representation of how motherhood requires you to be resourceful lol REAL LIFE," one commenter accurately wrote.
"Funny thing about the respiratory system, its located in my vagina," added another.
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This morning.
Mark Tighe tweetz:
Came across some awful driving this morning as this taxi man crushed the front wheel of a woman's bike as he drove into the cycle lane at a busy junction. Thankfully the woman was not injured. Gardaí were called….
Free the lanes.
Had a near miss last week as #taxi driver cut in tight 2 the kerb in front of me on Redmond Hill. He was most unapologetic #allocate4cycling
— Dr. Bríd McGrath (@BridMcGrath) March 8, 2019


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Cyber expert Jarno Vanto joins Crowell & Moring in NYC
Kansas City-headquartered Polsinelli has witnessed the departure of cybersecurity and privacy partner Jarno Vanto to Crowell & Moring in New York.
Prior to joining Polsinelli in 2017, Vanto worked at Helsinki-headquartered Borenius for over four years and also had a spell at Morrison & Foerster (MoFo).
With knowledge of United States and European Union privacy regulations, Vanto counsels' clients on technology transfer agreements, domestic and cross-border intellectual property licensing, as well as cybersecurity and privacy compliance.
His clients include Fortune 50 companies and start-ups which provide data-as-a-service, software-as-a-service, financial services, as well as healthcare and connected vehicle technologies.
New York managing partner Glen McGorty commented in a statement: "[Vanto] is a top technology lawyer with the legal and business insight to guide companies through data privacy concerns as they grow in the US market."
Vanto added that the firm is "growing significantly in Europe, a jurisdiction that is important to my practice and my clients," and he looks forward to "counselling clients on their privacy and data security needs, especially on GDPR and CCPA compliance".
Crowell & Moring is following the lead of rival US firms to invest in the growing cybersecurity and privacy market, with MoFo recruiting privacy lawyer Kristen Mathews from Proskauer Rose in March.Tagged with: · #IP · #technology · #financial · #Cyber risk · #Cybersecurity · #Privacy · #GDPR


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