text:
Q: ipvsadm output processing I need to process ipvsadm output which is similiar to this:
IP Virtual Server version 0.2.12 (size=16384)
Prot LocalAddress:Port Scheduler Flags
  -> RemoteAddress:Port             Forward Weight ActiveConn InActConn
TCP  lvs2.mack.net:0 rr persistent 360
  -> RS2.mack.net:0                 Route   1      0          0
  -> RS1.mack.net:0                 Route   1      0          0
  -> RS3.mack.net:0                 Route   1      0          0
TCP  lvs2.mack.net:https rr
  -> RS2.mack.net:https            Route   1      0          0
TCP  lvs2.mack.net:telnet rr
  -> RS2.mack.net:telnet            Route   1      0          0
  -> RS1.mack.net:telnet            Route   1      0          0
TCP  lvs2.mack.net:ssh rr
TCP  lvs2.mack.net:hvr rr

and on output I expect only these VIPs which have less than 2 real nodes 'alive' at the moment. So processing of example data above, expected output is:
TCP  lvs2.mack.net:https rr
  -> RS2.mack.net:https            Route   1      0          0
TCP  lvs2.mack.net:ssh rr
TCP  lvs2.mack.net:hvr rr

because as we see one VIP has only 1 real node active and the other two none avaialble.

A: Awk solution:
awk 'NR<4{ next }
     /TCP/{ if (host && c<2) printf "%s%s\n",host,(c==1? ORS node:""); host=$0; c=0 }
     host && $1=="->"{ node=$0; c++ }
     END{ if (host && c<2) print host }' file

*

*NR<4{ next } - skip the first 3 lines (NR indicates total number of rows been read)

*/TCP/ - on encountering host line started with TCP:

*

*host=$0 - capture the whole line into variable host

*c=0 - count for number of nodes for the current host

*if (host && c<2) printf "%s%s\n",host,(c==1? ORS node:"") - if there's a previously processed host and the number of its nodes is c<2 - print the host line and its node(if exists)

*host && $1=="->"{ node=$0; c++ } - for the currently processed host - capture its node line into variable node and incrementing the number of nodes c++

*END{ if (host && c<2) print host } - check for remaining host that has been processed but not printed

The output:
TCP  lvs2.mack.net:https rr
  -> RS2.mack.net:https            Route   1      0          0
TCP  lvs2.mack.net:ssh rr
TCP  lvs2.mack.net:hvr rr


text:
Cyworld digital music award (DMA) to T-ara
T-ara has won a Cyworld digital music award (DMA) for the 2nd consecutive time.
T-ara has won an award at DMA for the first time with their debut song 'Lie' and then for this month, they won again with the song 'TTL' a project with Supernova.
For the 38th DMA, which is for the month of August, T-ara won the 'Rookie of the Month' with their debut song 'Lie' and the for the 39th DMA, they won 'ting's Choice Artist' award for the song 'TTL' together with Supernova. 'ting's Choice Artist' is based on the Cyworld background music chart.
'TTL' is not only doing well up on Cyworld music chart, it is also doing well on other music charts like on Mnet, Melon, Bugs etc. The project group recently released 'TTL Listen 2' and will be expected to gain much love with the new version of the song.

redpajama_set_name: RedPajamaCommonCrawl


text:
Welcome To The Friendly City
In The Heart Of The Redwoods
July 12th - 18th, 2021
Bullfighters Only! / Quadiators
Bulls, Broncs, Bands & Brews
Jr. Rodeo
HISTORY, HERITAGE & HONOR
100 YEARS STRONG!
THE RODEO STARTS AT 1:30 PM SATURDAY JULY 17th, sponsored by Lithia Chrysler Dodge!
$10 Adults, $5 under 12! GET TICKETS FOR THE SATURDAY RODEO HERE!
AND 1:30PM SUNDAY THE 18th, sponsored by Humboldt Beer Distributors
$10 Adults, $5 under 12! GET TICKETS FOR THE SUNDAY RODEO HERE!
NOTE: Tickets for all events will be available at the gate, cash only, with an ATM on site!
FORTUNA RODEO PHOTOS BY CAROL NILES PHOTOGRAPHY:
RODEO EVENTS:
Breakaway Roping
This fast-paced event is the purest test of a cowgirl's lasso skills. As in steer wrestling, a calf leaves the shute at a run and after a head start trips the barrier holding back the cowgirl. The contestant then gives chase and attemps to lasso the calf's head. She has the rope tied to her saddle horn with a string and halts her horse. Time is called when the calf, which is still running, breaks the string.
Tie-Down Roping
Tie-down roping was derived from the practical method cowboys use to gather cattle for medical treatments or branding. The pursuit doesn't begin until the calf reaches its head start and triggers a trip lever releasing the barrier in front of the roper's chute. In seconds, the roper and horse will go from standstill to full-tilt gallop, racing after the calf and against the clock. Once in position, the rider ropes the calf, dismounts and runs to it. Meanwhile, the horse is trained to continually move backward to maintain tension on the rope. The cowboy then flanks the calf to its side, and with the use of a pigging string, ties up any 3 of the calf's legs. The clock stops when the cowboy throws both hands in the air. The cowboy then remounts his horse and rides forward, the calf must remain tied for six seconds. A 10 second penalty is added to that time if the cowboy and his horse break the barrier, which can be costly, where contestants are separated by fractions of a second.
Bareback Riding
The cowboy must rely on his strength and technique, with one hand in the air while the other hand desperately holds on to the rigging. Once out of the chute, the rider must keep his feet above the horse's shoulder before its front hooves hit the arena dirt on the first jump. This is called "marking out." Failure to do so results in a "NO Score". The cowboy should continually spur in rhythm with the horse's bucking to maintain stability and execute proper form. Judges consider both the rider and the horse in the score, looking at the horse's bucking action and the cowboy's spurring technique. The judges also look for the rider's willingness to lean far back on the horse and adjust to its wild action
Team Roping
Two riders on horseback are positioned on either side of the roping chute in preparation for the wild chase. After the steer gains a head start, the first roper, known as the header, must rope the steer in one of three ways: around both horns, around one horn and the head, or around the neck. If the header ropes the steer in any other fashion, then the team is disqualified. After the header ropes the steer and quickly dallies his lasso, he must quickly turn the steer to the left to set up his partner, the heeler. The heeler's task is to lasso both of the steer's hind legs; time is called when both horses turn to face each other with no slack in the ropes holding the steer. The riders are penalized 10 seconds if the header leaves the chute too soon and five seconds if the heeler only ropes one hind leg.
Saddle Bronc Riding
Rodeo's classic event, saddle bronc riding, evolved from the Old West when gritty cowboys fought to tame nature by breaking and training wild horses for their cattle ranches. A rider can easily receive a "No Score" for simply missing his "mark out," which requires both heels to touch the horse's shoulders on the first jump out of the chute. The rider also can be disqualified if he touches any part of the horse or his body with his free hand at any time during his ride. For eight seconds, the dirt arena becomes a dance floor as the rider uses strength and agility to spur in rhythm with the horse's bucking motion. To garner the best score, a cowboy must keep his toes turned outward, spur consistently from shoulder to saddle, and maintain balance and control of the horse. The horse's bucking action also is judged and calculated into the overall score. The Cowboy is judged 0-50 pts on his mechanics and the horse is judged 0-50 on its wildness and ability to buck.
Steer Wrestling
In steer wrestling, a cowboy uses only his strength and skills to tackle a runaway steer at least twice his size and weight. Steer wrestling is the fastest rodeo event, with a world record of 2.4 seconds The steer wrestler starts behind the barrier on one side of the steer, which is waiting to run in the chute, while a second cowboy on horseback, called a "hazer", waits on the opposite side. The hazer acts like a barrier preventing the steer from veering away from the bulldogger after it leaves the chute. Once the steer is given a head start, the bulldogger and hazer take off blocking either side of the 500-pound animal. The bulldogger slides off his horse, wraps his arms around the steer's horns and uses his whole body and strength to flip the steer on its side with all four legs pointing in the same direction. If the cowboy breaks the barrier too soon, he receives a 10 second penalty.
Barrel Racing
This all-women's rodeo event is a display of fine horsemanship as the rider and horse maneuver through a course racing against the clock. Timing is truly everything in barrel racing as a contestant's run is measured in hundredths of a second. Entering the arena at full speed, the horse and rider race to complete a cloverleaf pattern around three barrels in a triangle formation before exiting through the entrance. Dirt flies as the horse and rider try to "hug the barrels" as close as possible to remove seconds from their time. The rider must be accurate or else they risk knocking over a barrel and adding a penalty of five precious seconds to their total time.
Bull riding is often considered the most dangerous sport in rodeo. While most rodeo events evolved from ranch hand practices, bull riding started from the fearless, wild character of cowboys brave enough to ride nature's horned beast. The fate of the ride is in the cowboy's own hand as he grips on to the bull rope. A bull rope is a flat-braided rope with a single hand-hold wrapped around the bull's chest and held fast by the cowboy's riding hand. A bull rider can be disqualified for touching the bull or himself with his free hand during the ride. A bull rider's challenge is an eight second ride; however time isn't the only factor in the score. As in all rough stock events, the bull's bucking efforts account for half of the rider's points. Each bull is unpredictable, dangerous and specific in its own bucking fashion. Some bulls spin continuously in a circle, or jerk side to side, and even jump in the air relentlessly kicking.
Fortuna Rodeo Sponsors:
See the full list of Fortuna Rodeo Sponsors HERE!
Site © 2021 FORTUNA RODEO ASSOCIATION.
Site powered by PRECISION INTERMEDIA.
Photos by CAROL NILES PHOTOGRAPHY and PRECISION INTERMEDIA.
Content from this site may not be reused without permission.


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Join us at the Rivers Club in Pittsburgh on June 6, 2019, at 12:00 p.m. for lunch with Pittsburgh sports icon, Bill Hillgrove! Registration opens at 11:30 a.m. Please note that the time has changed.
Celebrate St. Anthony School Programs' mission to educate children ages 5-21 with Down Syndrome, Autism Spectrum Disorder and other Intellectual Disabilities at the Spirit of St. Anthony Luncheon.
Our 2019 keynote speaker is Bill Hillgrove, the voice of the Pittsburgh Steelers and University of Pittsburgh Panthers. He will share stories of faith, his long history with St. Anthony's and life lessons learned throughout his career.
Advance reservations are required. Tickets are $50 per seat or $400 for a table of eight. For more information about this year's event or to reserve your seats, please contact Heather Schmuck, Special Events Coordinator, at 724-940-9020, ext. 104 or via email.
Please join us as we celebrate St. Anthony School Programs.
Tickets are $50 per seat or $400 for a table of eight.


text:
Intracellular Serine Protease Inhibitor SERPINB4 Inhibits Granzyme M-Induced Cell Death
Pieter J. A. de Koning,
Current address: Division of Surgical Specialties, University Medical Center Utrecht, Utrecht, The Netherlands
Affiliation Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands
J. Alain Kummer,
Current address: Department of Pathology, St. Antonius Hospital, Nieuwegein, The Netherlands
Stefanie A. H. de Poot,
Razi Quadir,
Roel Broekhuizen,
Anne F. McGettrick,
Current address: School of Biochemistry and Immunology, Trinity College Dublin, Dublin, Ireland
Affiliation School of Biomolecular and Biochemical Science, University College Dublin, Dublin, Ireland
Wayne J. Higgins,
Bart Devreese,
Affiliation Laboratory for Protein Biochemistry and Biomolecular Engineering, Department of Biochemistry and Microbiology, Ghent University, Ghent, Belgium
D. Margaret Worrall,
Niels Bovenschen
* E-mail: n.bovenschen@umcutrecht.nl
Pieter J. A. de Koning J. Alain Kummer ... Niels Bovenschen
Granzyme-mediated cell death is the major pathway for cytotoxic lymphocytes to kill virus-infected and tumor cells. In humans, five different granzymes (i.e. GrA, GrB, GrH, GrK, and GrM) are known that all induce cell death. Expression of intracellular serine protease inhibitors (serpins) is one of the mechanisms by which tumor cells evade cytotoxic lymphocyte-mediated killing. Intracellular expression of SERPINB9 by tumor cells renders them resistant to GrB-induced apoptosis. In contrast to GrB, however, no physiological intracellular inhibitors are known for the other four human granzymes. In the present study, we show that SERPINB4 formed a typical serpin-protease SDS-stable complex with both recombinant and native human GrM. Mutation of the P2-P1-P1′ triplet in the SERPINB4 reactive center loop completely abolished complex formation with GrM and N-terminal sequencing revealed that GrM cleaves SERPINB4 after P1-Leu. SERPINB4 inhibited GrM activity with a stoichiometry of inhibition of 1.6 and an apparent second order rate constant of 1.3×104 M−1s−1. SERPINB4 abolished cleavage of the macromolecular GrM substrates α-tubulin and nucleophosmin. Overexpression of SERPINB4 in tumor cells inhibited recombinant GrM-induced as well as NK cell-mediated cell death and this inhibition depended on the reactive center loop of the serpin. As SERPINB4 is highly expressed by squamous cell carcinomas, our results may represent a novel mechanism by which these tumor cells evade cytotoxic lymphocyte-induced GrM-mediated cell death.
Citation: de Koning PJA, Kummer JA, de Poot SAH, Quadir R, Broekhuizen R, McGettrick AF, et al. (2011) Intracellular Serine Protease Inhibitor SERPINB4 Inhibits Granzyme M-Induced Cell Death. PLoS ONE 6(8): e22645. https://doi.org/10.1371/journal.pone.0022645
Editor: Ashley M. Buckle, Monash University, Australia
Received: January 19, 2011; Accepted: June 30, 2011; Published: August 3, 2011
Copyright: © 2011 de Koning et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by the Dutch Cancer Society (KWF) (grant UU-2004-3047 to JAK and grant UU-2009-4302 to NB and JAK) and the Netherlands Organization for Scientific Research (NWO) (grant 916.66.044 to NB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells (i.e. cytotoxic lymphocytes) play a pivotal role in the effector arm of the immune response that eliminate virus-infected cells and tumor cells [1]. Cytotoxic lymphocytes predominantly destroy their target cells by releasing the content of their cytolytic granules. These granules contain perforin and a family of unique structurally homologous serine proteases known as granzymes [2]. While perforin facilitates the entry of granzymes into the target cell, the latter induce cell death by cleaving critical intracellular substrates [3].
In humans, five different granzymes (GrA, GrB, GrH, GrK, and GrM) are known that differ on the basis of their substrate specificity [4]. All granzymes induce cell death with partially overlapping morphological hallmarks [4]. While GrA and GrB have been extensively studied, far less is known about the molecular cell death mechanisms of the other human granzymes [5]. Recently, it has been demonstrated that GrM, which is highly expressed by NK cells, NKT cells, γδ-T cells, and CD8+ effector T cells [6], [7], [8], mediates a major and novel perforin-dependent cell death pathway that plays a significant role in cytotoxic lymphocyte induced death [9]. In tumor cell lines, GrM directly and efficiently cleaves a diverse set of substrates, i.e. ICAD, PARP, HSP75, ezrin, α-tubulin, PAK 2, survivin, and nucleophosmin [10], [11], [12], [13], [14].
Tumor cells can escape from cytotoxic lymphocyte-induced killing by expression of cell death inhibitors in their cytoplasm, like the caspase-inhibitors XIAP and FLIP [15], [16], and the GrB-inhibitor SERPINB9 (PI9) [17]. SERPINB9 is the only known intracellular human granzyme inhibitor and protects against GrB-induced apoptosis [17], [18]. Expression of SERPINB9 is associated with a poor clinical outcome in various types of tumors (e.g. lymphomas and melanomas) [19], [20], [21]. SERPINB9 belongs to the intracellular (B-clade) sub-family of human serine protease inhibitors (serpins). Serpins share a unique inhibitory mechanism. Upon cleavage by a specific target protease in their reactive center loop (RCL), serpins undergo a conformational change after which the serpin and the target protease are covalently bound, leaving the latter kinetically inactive [22].
In contrast to GrB, no physiological intracellular inhibitors are known for the other four human granzymes [23]. Since GrM is a very potent specialized inducer of tumor cell death [5], [24] and plays an important role in anti-tumor function in vivo [25], we aimed to identify an intracellular inhibitor of human GrM. In the present study, we demonstrate that SERPINB4 [also called squamous cell carcinoma antigen 2 (SCCA-2) or leupin] directly inhibits human GrM proteolytic activity and that overexpression of SERPINB4 in HeLa cells inhibits recombinant GrM-induced as well as NK cell-mediated cell death. This may represents a novel mechanism by which tumor cells evade GrM-mediated killing by cytotoxic lymphocytes.
Expression and purification of recombinant human GrM and the catalytically inactive GrM-SA variant was performed as described previously [10]. Briefly, cDNA encoding mature human GrM (residues Ile26-Ala257) was cloned into the yeast expression vector pPIC9 (Invitrogen, Paisley, UK). Catalytically inactive GrM-SA, in which the Ser195 residue in the catalytic center is replaced by Ala, was generated by site-directed mutagenesis (Stratagene, Cedar Creek, TX). Plasmids were transformed into the GS115 strain of P. pastoris (Invitrogen) and granzymes were expressed in conditioned media for 72 h. Recombinant GrM and GrM-SA were purified to homogeneity by cation-exchange chromatography (GE Healthcare, Diegem, Belgium) and dialyzed against 50 mM Tris (pH 7.4) and 150 mM NaCl. Recombinant GrM, but not GrM-SA, was active as determined by a synthetic chromogenic leucine substrate (Bachem, Weil am Rhein, Germany).
Expression and purification of recombinant SERPINB4 wild type and SERPINB4 RCL-mutant was performed using the expression vector pRSETC (Invitrogen) as described previously [26]. SERPINB4, coding an N-terminal His6-tagged fusion protein, and SERPINB4 RCL-mutant, in which the P2-Glu353, P1-Leu354, P1′-Ser355 amino acids were mutated into P2-Gln353, P1-Gly354, P1′-Ala355, were expressed in E.coli BL21 (DE3) using Overnight Express auto inducing medium (Merck, Nottingham, UK) containing 100 µg/ml ampicillin. Following growth at 37°C for 24 h, cells were harvested by centrifugation at 15,000 g for 30 minutes and lysed using Bugbuster (Merck) lysis reagent. Soluble material was clarified by centrifugation of the lysate at 21,000 g for 30 minutes at 4°C. The recombinant serpin was purified using a His-Bind Purification kit (Merck). Pooled imidazole eluted fractions were buffer exchanged into 50 mM Tris pH 8.0 and recombinant protein was stored at −80°C until required. Concentrations of the purified proteins were measured according to the procedure of Bradford (Bio-Rad, Hercules, CA).
Generation of a novel mAb against human GrM
Mice were immunized with purified recombinant human GrM. Obtained hybridomas were screened for antibodies that reacted with GrM in different applications as previously described [27]. Anti-GrM mAb 3D4D7 (IgG1 isotype) was highly sensitive against Western-blotted recombinant GrM as well as native GrM from NK cell lysates (Fig. 1). Cross-reactivity of mAb 3D4D7 against recombinant human GrB and GrK was excluded (data not shown).
Figure 1. SERPINB4 forms a typical serpin-protease SDS-stable complex with recombinant and native human GrM.
(A) Purified recombinant GrM (1.8 µM) and SERPINB4 (1.8 µM) were incubated for 1 h at 37°C. All samples were treated with PNGase F, separated by SDS-PAGE, and analyzed by SimplyBlue staining. (B) Cell lysate of the NK cell line KHYG-1 (33 µg) was incubated with recombinant SERPINB4 (0, 50, 150, and 450 ng) for 2 h at 37°C. Subsequently, samples were immunoblotted for native GrM. GrM, SERPINB4, GrM-SERPINB4 complexes, and cleaved SERPINB4 (SERPINB4*) are indicated.
Cell culture and transfection
293T and Jurkat cells were maintained in DMEM and RPMI-1640 medium (Invitrogen), respectively, supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Sigma), 100 U/ml penicillin, and 100 µg/ml streptomycin (Invitrogen). The human NK cell line KHYG-1 [28] was purchased from the Health Science Research Resources Bank (JCRB0156) of the Japan Health Sciences Foundation and cultured similarly to Jurkat cells, with the addition of 50 ng/ml recombinant human interleukin 2 (Wako, Osaka, Japan). The previously described HeLa cell lines, stably transfected with pcDNA3 SERPINB4, pcDNA3 SERPINB4 RCL-mutant, or pcDNA3 vector only [26], were cultured similarly to 293T cells, with the addition of G418 (0.5 mg/ml). The plasmid pEGFP-N1 containing C-terminal green fluorescent protein (GFP)-tagged SERPINB4 (kindly provided by Dr. Wun-Shaing W. Chang, National Institute of Cancer Research, National Health Research Institutes, Taiwan, ROC) or mock control vector were transiently transfected into 293T cells using linear polyethylenimine (PEI) (Polysciences, Warrington, PA), according to the manufacturer's instructions. Cell lysates were prepared by three freezing-thawing cycles in liquid nitrogen of cells resuspended in a buffer containing 20 mM Tris (pH 7.4) and 150 mM NaCl. Protein concentrations of the supernatants were measured according to the procedure of Bradford.
Analysis of complex formation by SDS-PAGE and immunoblotting
Indicated amounts of purified recombinant SERPINB4 and GrM proteins were incubated in 20 mM Tris (pH 7.4) and 150 mM NaCl for 1 h at 37°C. When noted, Jurkat or KHYG-1 cell lysate was incubated with the recombinant protein(s) for the indicated times at 37°C. Samples containing recombinant proteins only were additionally treated with PNGase F (New England BioLabs, Ipswich, MA), according to the manufacturer's instructions. Subsequently, samples were separated on 10% SDS-PAGE gels under reducing conditions. For SDS-PAGE analysis, gels were stained with SimplyBlue (Invitrogen), according to the manufacturer's instructions. For immunoblot analysis, proteins were transferred onto immobilon–P membranes (Millipore, Billerica, MA). After blocking with 5% (w/v) Marvel dried skimmed milk (Premier International Foods, Coolock, UK) in TBS-T (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% Tween-20), membranes were incubated for two hours at RT with our novel mouse anti-human GrM mAb (clone 3D4D7; 1.0 µg/ml), mouse anti-human α-tubulin mAb (clone B-5-1-2; 0.4 µg/ml) (Sigma), mouse anti-human nucleophosmin (clone FC-61991; 2.0 µg/ml) (Invitrogen), rabbit anti-human nm23H-1 (clone C-20:sc-343; 1.0 µg/ml) (Santa Cruz Biotechnology, Santa Cruz, CA), or mouse anti-human β-actin mAb (clone 2A2.1) (US Biological, Swampscott, MA). Goat anti-mouse IgG+IgM HRP (Biosource, Camarillo, CA) conjugate was used as secondary antibody. Bound antibodies were visualized using 3,3′-diaminobenzidine (DAB) (0.6 mg/ml) or enhanced chemiluminescence substrate (ECL) (GE Healthcare).
Cell lysates of mock and GFP-tagged SERPINB4 transfected 293T cells were incubated with 10 µg/ml anti-GFP mAb (Roche, Mannheim, Germany) and protein A/G plus-agarose (Santa Cruz Biotechnology), and rotated end-over-end in non-sticky microfuge tubes (Ambion, Austin, TX) for 16 h at 4°C. Precipitates were washed three times with 50 mM Tris (pH 7.4), 150 mM NaCl and incubated with 2.3 µM rh-GrM for 1 h at 37°C. Next, the samples were washed twice more and finally boiled in reducing Laemmli sample buffer. Immunoblotted samples were stained by Coomassie Brilliant Blue (BioRad) and the cleavage-product of interest was excised and identified by Edman degradation, using a 476A protein sequencer (Applied Biosystems, Foster City, CA).
Stoichiometry of inhibition
Increasing concentrations (0–3 µM) of recombinant SERPINB4 was incubated with a constant amount of recombinant human GrM (2 µM) in 50 mM Tris (pH 7.4) and 150 mM NaCl for 2 h at 37°C. Samples were diluted 10-fold in 100 mM Tris (pH 7.4), 200 mM NaCl, and 0.01% Tween containing 1 mM synthetic chromogenic leucine substrate (AAPL-pNA) (Bachem) to terminate the inhibition reactions and transferred to a microplate (Greiner, Kremsmunster, Austria) to assay residual GrM activity. The velocity of substrate hydrolysis by residual active GrM was measured at A405 using a microtiter plate reader (Anthos, Cambridge, UK). The fractional activity (velocity of GrM with a serpin/velocity of GrM only) was plotted against the ratio of [serpin]0/[GrM]0. Linear regression analysis was used to determine the x-axis intercept as a value for the stoichiometry of inhibition.
Apparent second order rate constant
The interaction of SERPINB4 with GrM was determined under second order conditions [29]. Recombinant GrM was incubated with recombinant SERPINB4 at equimolar concentrations. Samples of this reaction were taken after 30, 60, 120, 180, 240 and 360 seconds and directly diluted 20-fold in 100 mM Tris (pH 7.4), 200 mM NaCl, and 0.01% Tween containing 1 mM of the chromogenic substrate AAPL-pNA. The rate of substrate hydrolysis by GrM was measured in time at A405 using a microtiter plate reader (Anthos). Since serpin-serine protease interactions are irreversible, velocities of the substrate hydrolysis were converted to residual active GrM concentrations using a GrM concentration standard curve incubated without SERPINB4. The rate of change in the amount of residual active GrM over time is described in equation (1), where the slope of the plot of reciprocal residual active GrM over time yields the apparent second order rate constant (kinh). (1)
RT-PCR and immunoblot analysis for SERPINB4 expression
RNA was isolated from (stably transfected) HeLa cells using RNA isolator™ (Genosys). RNA (1–5 µg) was reverse transcribed using MMLV-reverse transcriptase, and the resulting cDNA was used for RT-PCR amplification. The following primers were used: GAPDH-sense 5′-TGAAGGTCGGAGTCAACG-3′, GAPDH-antisense 5′-CATGTGGGCCATGAGGTC-3′, SERPINB4-sense 5′-GGGGGATCCATATGAATTCACTCAGTGAAG-3′, and SERPINB4-antisense 5′-CCCCCGGGTACCTACGGGGATGAGAATCTG-3′. Immunoblot analysis for SERPINB4 protein expression by (stably transfected) HeLa cells was performed as previously described [26].
Granzyme M killing assays
HeLa cells stably transfected with SERPINB4 or SERPINB4 RCL-mutant were grown to confluence in a 96-well tissue-culture plate. Cells were washed twice in serum-free DMEM, after which they were incubated at 37°C with a sublytic dose of streptolysin O (SLO) (0.5 µg/ml) (Sigma) and indicated concentrations of granzyme in serum-free DMEM for 30 min. Cells were washed twice with supplemented DMEM, after which cells were incubated for indicated periods of time at 37°C. For the methylene blue assay, cells were carefully washed with PBS and remaining adherent cells were fixed with methanol for 30 min at room temperature. After drying, cells were stained with 0.05% (w/v) methylene blue dye (Merck) in borate-buffered saline (pH 8.4) for 15 min, washed 3 times with water, and air dried. Bound dye was dissolved in 0.1 N HCl (100 µl/well) and measured photospectrometrically at 630 nm. To assess cell viability by flow cytometry, cells were stained with AnnexinV-fluos (Invitrogen) and propidium iodide (PI) for 15 min in a buffer containing 140 mM NaCl, 4 mM KCl, 0.75 mM MgCl2, 1.5 mM CaCl2, and 10 mM HEPES (pH 7.4). Flow cytometry was performed on a FACSCalibur (BD Biosciences) and data was analyzed using CellQuest Pro software (BD Biosciences).
NK-cell mediated cytotoxicity assay
HeLa cells stably transfected with SERPINB4 or SERPINB4 RCL-mutant were grown to confluence in a 96-well tissue-culture plate and loaded for 10 min with 10 µM of the fluorescent cell staining dye carboxyfluorescein diacetate succinimidyl ester (CFDA-SE). These HeLa cells were co-cultured with KHYG-1 NK cells in varying E∶T ratio's for 16 h at 37°C. Cells were stained with PI and analyzed by flow cytometry. During data analysis, a gate on CFDA-SE positive cells was used to distinguish between KHYG-1 and HeLa cells. The percentage of living untreated HeLa cells was set at 100% and the percentages of treated cells were matched correspondingly, after which the percentage of specific cytotoxicity was calculated.
All statistical analyses were performed using the Student's t-test.
SERPINB4 forms a typical serpin-protease SDS-stable complex with human GrM
Upon cleavage by a specific target protease, most serpins form SDS-stable covalent-bound complexes with that protease, in which the latter one becomes inactive [22]. Since human GrM displays a restricted cleavage site specificity in that it predominantly cleaves after P1-Leu [10], [11], [12], [30] and that SERPINB4 is the only human intracellular B-clade serpin that harbours a Leu at the putative P1-position in its RCL [31], [32], we investigated complex formation of SERPINB4 with GrM by SDS-PAGE analysis using purified recombinant proteins (Fig. 1A). Purified recombinant GrM and SERPINB4 migrated with a molecular mass of 28 and 44 kDa, respectively (Fig. 1A, left and right lane). Incubation of GrM with SERPINB4 revealed an SDS-stable complex band with the expected molecular mass of around 70 kDa (Fig. 1A, middle lane). The majority of SERPINB4 molecules formed a complex with GrM, whereas a small fraction of SERPINB4 was cleaved by GrM without trapping this protease. Next, we studied whether SERPINB4 could also bind to and form an SDS-stable complex with native human GrM (Fig. 1B). The human NK cell line KHYG-1 was used as a source for native GrM. Incubation of a KHYG-1 cell lysate with recombinant SERPINB4 and subsequent immunoblotting for the protease revealed the formation of a complex between native GrM and recombinant SERPINB4 in a concentration-dependent manner (Fig. 1B). These data demonstrate that SERPINB4 forms a classical SDS-stable serpin-protease complex with both recombinant and native human GrM.
GrM cleaves SERPINB4 in its RCL after P1-Leu
To investigate if complex formation of SERPINB4 with GrM depends on the RCL and corresponding conformational change of the serpin, a RCL-mutant of SERPINB4 was employed in which the amino acids at the putative P2(Glu353)-P1(Leu354)-P1′(Ser355) positions were mutated into P2(Gln353)-P1(Gly354)-P1′(Ala355). Western blot analysis for GrM revealed a complex with a molecular mass of about 70 kDa when the purified recombinant wild type proteins of GrM and SERPINB4 were incubated (Fig. 2A). In contrast, no complex formation was detected upon incubation of SERPINB4 RCL-mutant with GrM. Furthermore, a mutated catalytically inactive counterpart of GrM was included (GrM-SA). As expected, this GrM-SA was not able to form a complex with SERPINB4 (Fig. 2A). Thus, complex formation of SERPINB4 with GrM appears to depend on the RCL of the serpin and the catalytic triad of the protease.
Figure 2. Mutation of the SERPINB4 RCL at the P2-P1-P1′ positions completely abolishes complex formation with human GrM.
(A) A RCL-mutant of SERPINB4 was employed in which the amino acids at the putative P2(Glu353)-P1(Leu354)-P1′(Ser355) positions were mutated into P2(Gln353)-P1(Gly354)-P1′(Ala355). Purified recombinant GrM (0.9 µM), GrM-SA (0.9 µM), SERPINB4 (0.9 µM), and SERPINB4 RCL-mutant (0.9 µM) were incubated for 1 h at 37°C. All samples were treated with PNGase F, separated by SDS-PAGE, and immunoblotted for GrM. Bound antibodies were visualized using DAB. (B) Cell lysates of 293T cells transfected with C-terminal GFP-conjugated SERPINB4 or an empty vector (mock) were incubated with recombinant GrM (0.5 µM) for 1 h at 37°C. Subsequently, samples were immunoblotted for GFP. SERPINB4-GFP represents the full length protein, whereas SERPINB4*-GFP depicts the C-terminal cleavage product. (C) Schematic representation of C-terminal GFP-conjugated SERPINB4, including the GrM cleavage site after Leu354 at the P1-position in the RCL.
Next, the cleavage site of GrM in the RCL of SERPINB4 was determined. Therefore, cell lysates of 293T cells expressing either C-terminal GFP-tagged full length SERPINB4 or an empty vector were incubated with GrM. Immunoblotting for GFP showed a C-terminal cleavage product of SERPINB4 (SERPINB4*-GFP) with the expected molecular mass of about 29 kDa (Fig. 2B), as the RCL of SERPINB4 is near its C-terminus and the molecular mass of the linked GFP is about 25 kDa. Immunoprecipitation of GFP-conjugated SERPINB4 from 293T cell lysate and N-terminal sequencing of the subsequent GrM-induced C-terminal cleavage fragment revealed that GrM indeed cleaves SERPINB4 in its RCL, at least after the amino acid Leu354 at the P1-position in the sequence 351VVEL↓SSPS358 (Fig. 2C).
Stoichiometry of inhibition of GrM and SERPINB4
The interaction of a serpin with its physiological target protease usually results in complex formation with a stoichiometry of inhibition (SI) value close to 1. The SI value indicates how many serpin-molecules are needed to inhibited one molecule of target protease. If a serpin is also a substrate in parallel to an inhibitor of its target protease, this is reflected by a SI value greater than 1. The SI value for inhibition of GrM by SERPINB4 was determined by measurement of residual GrM activity at increasing [SERPINB4]0/[GrM]0 ratios (Fig. 3A). Complete inhibition of GrM activity was obtained at a [SERPINB4]0/[GrM]0 ratio of 1.6±0.07 (mean ± SD of 4 independent experiments) (Fig. 3A). This indicates that about three molecules of SERPINB4 are needed to inhibit two GrM molecules.
Figure 3. Kinetic analyses of GrM-inhibition by SERPINB4.
(A) Purified recombinant human GrM (2 µM) was incubated with different concentrations of recombinant SERPINB4 (0–3 µM) for 2 h at 37°C. Residual GrM activity was monitored by addition of a synthetic chromogenic leucine substrate (1 mM) and measuring A405 in time. The fractional activity (velocity of substrate hydrolysis by GrM in the presence of SERPINB4/velocity of substrate hydrolysis by GrM without SERPINB4) was plotted against the ratio of [SERPINB4]0/[GrM]0. Linear regression analysis was used to calculate the x-intercept as a value for the SI and determined to be 1.6±0.07 (mean ± SD of 4 independent experiments). (B) The apparent second order rate constant (kinh) of the inhibition of GrM by SERPINB4 was determined under second order conditions. Equimolar concentrations of recombinant human GrM and SERPINB4 were incubated. Aliquots were removed at different time points and the reaction was directly stopped by 20-fold dilution with buffer containing a synthetic chromogenic leucine substrate (1 mM). Residual GrM activity was determined by measuring the velocity of substrate hydrolysis in time at A405. These velocities were converted to residual active GrM concentrations using a GrM concentration standard curve. The apparent second order rate constant (kinh) was calculated from the slope of the plot of reciprocal residual active GrM over time and determined to be 1.3×104 M−1s−1 (graph indicates one representative example of three independent experiments with similar results).
Kinetic analysis of the inhibition of GrM activity by SERPINB4
To determine the rate of complex formation between GrM and SERPINB4, the apparent second order rate constant (kinh) was measured under second order conditions. Upon incubation of recombinant GrM with equimolar concentrations of SERPINB4 samples were taken in time to measure the residual GrM-activity. Addition of a synthetic chromogenic leucine substrate to these aliquots revealed that the concentration of active GrM decreased linear in time (Fig. 3B). The apparent second order rate constant (kinh) was calculated from the slope of the plot of reciprocal residual active GrM over time and was determined to be 1.3 (±0.05)×104 M−1s−1 (mean ± SD of 3 independent experiments). This kinh was not affected by inclusion of the glycosaminoglycan heparin in these experiments, which is a known co-factor of several serpins that enhances the kinh towards their target proteases [33] (data not shown).
SERPINB4 inhibits cleavage of natural macromolecular substrates by GrM
The inhibitory effect of SERPINB4 on the activity of human GrM was further investigated using natural macromolecular substrates of GrM. GrM and SERPINB4 were incubated at various concentration-ratios and GrM-activity towards two known direct GrM-substrates [10], [11] in a Jurkat tumor cell lysate was determined by immunoblotting (Fig. 4). As expected, in the absence of SERPINB4, human recombinant GrM efficiently cleaved the 55 kDa α-tubulin subunit as well as the 37 kDa nucleolar phosphoprotein nucleophosmin. Recombinant SERPINB4 impaired cleavage of both α-tubulin and nucleophosmin by GrM in a concentration-dependent manner (Fig. 4). Excessive concentrations of SERPINB4 over GrM completely inhibited GrM activity towards α-tubulin, whereas some nucleophosmin cleavage was still observed, suggesting that nucleophosmin is a more efficient GrM-substrate than α-tubulin. β-actin served as a loading control (Fig. 4). In conclusion, SERPINB4 inhibits GrM-mediated cleavage of the natural macromolecular substrates α-tubulin and nucleophosmin in a tumor cell lysate.
Figure 4. SERPINB4 inhibits GrM-mediated cleavage of macromolecular substrates.
Indicated concentrations of recombinant GrM and SERPINB4 were incubated for 2 h at 37°C. Jurkat tumor cell lysate (2 µg) was added to the samples and incubated for another 4 h at 37°C to determine the residual GrM-activity towards macromolecular substrates. Finally, all samples were immunoblotted for α-tubulin, nucleophosmin, and β-actin.
Overexpression of SERPINB4 inhibits recombinant GrM-induced as well as NK cell-mediated cell death
To examine whether SERPINB4 also inhibits the proteolytic activity of GrM in tumor cells, the effect of overexpression of SERPINB4 in HeLa cells on GrM-induced cell death was investigated. First, SERPINB4 RNA and protein expression by HeLa cells stable transfected with SERPINB4, SERPINB4 RCL-mutant, or an empty vector were determined. RT-PCR analysis revealed SERPINB4 RNA expression in both HeLa cells that overexpressed SERPINB4 and SERPINB4 RCL-mutant (Fig. 5A, upper panel). Mock-transfected HeLa cells and HeLa cells transfected with the empty vector did not express SERPINB4 RNA. GAPDH RNA expression served as a control for RNA/cDNA input (Fig. 5A, lower panel). In accordance with the RNA expression, immunoblot analysis revealed SERPINB4 protein expression by both HeLa cells stable transfected with SERPINB4 and SERPINB4 RCL-mutant, and not by mock-transfected HeLa cells or HeLa cells transfected with the empty vector (Fig. 5B). Second, a GrM cell death assay was employed in which the pore-forming protein SLO was used to deliver recombinant GrM into HeLa cells that overexpressed either SERPINB4 or SERPINB4 RCL-mutant. As a control, we included the catalytically inactive GrM mutant (i.e. GrM-SA). The methylene blue assay revealed that treatment of the cells with only a sublytic dose of SLO resulted in about 20% reduction of viable cells. As expected, the percentage of viable HeLa cells that overexpressed inactive SERPINB4 RCL-mutant significantly decreased upon treatment with SLO and recombinant GrM, but not with recombinant GrM only, SLO only, or the SLO/GrM-SA combination (Fig. 5C, left panel). In contrast, GrM-induced cell death of HeLa cells was fully rescued by overexpression of SERPINB4 (Fig. 5C, right panel). Similar results were obtained on a single cell level by staining with Annexin V and PI and subsequently FACS analysis. Again, GrM-induced cell death was inhibited by overexpression of SERPINB4 (Fig. 5D).
Figure 5. Overexpression of SERPINB4 in HeLa cells inhibits GrM-induced cell death.
(A) RT-PCR analysis of stably transfected HeLa cells for SERPINB4 and GAPDH mRNA expression (upper and lower panel, respectively). (B) Immunoblot analysis of SERPINB4 protein expression by HeLa cells stably transfected with pcDNA3 SERPINB4, pcDNA3 SERPINB4 RCL-mutant or pcDNA3 empty vector. (C) HeLa cells stably transfected with SERPINB4 or SERPINB4 RCL-mutant were treated with the indicated combinations of a sublytic dose of SLO (500 ng/ml), recombinant GrM (0.5 µM), and/or recombinant GrM-SA (0.5 µM) for 16 h at 37°C. Viable cells were quantified using the methylene blue assay. Data represent the percentages of viable cells as compared to HeLa cells that overexpressed SERPINB4 RCL-mutant and were treated with buffer only, which was set as 100%. Figure represents the mean ± SD of 4 independent experiments; * p<0.05. (D) HeLa cells stably transfected with SERPINB4 or SERPINB4 RCL-mutant were treated with the indicated combinations of a sublytic dose of SLO (500 ng/ml), recombinant GrM (1 µM), and/or recombinant GrM-SA (1 µM) for 20 h at 37°C. Cell viability was determined using flow cytometry, with AnnexinV and PI negative cells considered viable (mean ± S.D., n = 3, ** p<0.005). (E) HeLa cells stably transfected with SERPINB4 or SERPINB4 RCL-mutant were treated with the indicated combinations of a sublytic dose of SLO (500 ng/ml), recombinant GrM (1 µM), and/or recombinant GrM-SA (1 µM) for 4 h at 37°C. Total cell lysates were immunoblotted using antibodies against nucleophosmin and nm23H-1 (which served as a loading control).
To further determine whether GrM is inactivated by SERPINB4 after intercellular delivery, cleavage of the preferred GrM-substrate nucleophosmin was investigated. As expected, nucleophosmin was cleaved upon SLO-mediated delivery of GrM, and not GrM-SA, into HeLa cells overexpressing SERPINB4 RCL mutant (Fig. 5D, right lanes). In contrast, GrM-mediated intracellular cleavage of nucleophosmin was inhibited by overexpression of SERPINB4 (Fig. 5D, left lanes), indicating that GrM is inactivated by SERPINB4 upon intracellular delivery.
In addition to exogenous recombinant GrM-induced cell death, we also investigated whether SERPINB4 inhibits endogenous native GrM-induced cell death. The NK cell line KHYG-1, which is known to express large amounts of GrM and hardly, if any, GrA and GrB [34], was co-cultured in various ratios with HeLa cells stably transfected with either SERPINB4 or SERPINB4 RCL mutant. HeLa cells were killed by KHYG-1 cells in a ratio dependent manner (Fig. 6). In contrast, overexpression of SERPINB4 significantly inhibited KHYG-1 mediated cell death as compared to HeLa cells stably transfected with SERPINB4 RCL mutant. These results indicate that cellular overexpression of SERPINB4 inhibits recombinant GrM-induced as well as NK cell-mediated cell death and that this inhibition is dependent on the RCL of the serpin.
Figure 6. Overexpression of SERPINB4 in HeLa cells inhibits NK cell-mediated cell death.
HeLa cells stably transfected with SERPINB4 or SERPINB4 RCL-mutant were loaded with the fluorescent cell staining dye CFDA-SE and co-cultured with KHYG-1 NK cells in varying E∶T ratio's for 16 h at 37°C. Cells were stained with PI and analyzed by flow cytometry. HeLa cells were separated from KHYG-1 cells by gating for CFDA-SE positive cells. Depicted is the percentage of specific cytotoxicity (mean ± SD, n = 3, * p<0.05, ** p<0.005).
The granule-exocytosis pathway is the major mechanism for cytotoxic lymphocytes to kill tumor and virus-infected cells [1], [2], [3], [4]. Tumor cells can evade GrB-mediated cell death by expression of the intracellular serine protease inhibitor SERPINB9 [17], [18]. However, such physiological intracellular inhibitors are unknown for the other four human granzymes. In mice, GrM is inhibited by the murine serpin SPI-CI, which protects cells from GrM-mediated cell death [35]. SPI-CI is unique for mice and no human orthologue is known. In the present study, we show for the first time that human GrM also has an endogenous intracellular inhibitor that protects cells from GrM-mediated killing. We have demonstrated that GrM cleaves SERPINB4 in its RCL after P1-Leu, resulting in the formation of a typical serpin-protease SDS-stable complex (Figs. 1, 2, 3). Furthermore, SERPINB4 inhibited the proteolytic activity of GrM towards natural macromolecular substrates (Fig. 4 and 5E) and overexpression of SERPINB4 in HeLa cells inhibited recombinant GrM-induced as well as NK-cell mediated cell death (Fig. 5 and 6).
Our findings are consistent with the facts that GrM preferably cleaves after a P1-Leu and, to a lesser extent, after P1-Met [30], [36], and that SERPINB4 harbours a Leu at the P1-position in its RCL [31], [37]. Indeed, mutation of the P2-P1-P1′ triplet in the SERPINB4 RCL completely abolished complex formation with GrM (Fig. 2A) and N-terminal sequencing revealed that GrM cleaved SERPINB4 at least after the Leu residue at the RCL P1-position (Fig. 2C). In addition, protection of HeLa cells against both recombinant GrM-induced and NK cell-mediated cell death by cellular overexpression of SERPINB4 also depended on the RCL of the serpin (Fig. 5 and 6). Although SERPINB4 is the only intracellular serpin that meets the primary specificity of human GrM in that it harbours a Leu or Met amino acid at the putative P1-position in its RCL [32], [38], we cannot fully exclude that other intracellular serpins than SERPINB4 inhibit human GrM. Few serpins are known to use more than one RCL P1-residue to inhibit different target proteases, thereby broadening their inhibitory profile [38]. Interestingly, the GrB-inhibitor SERPINB9 harbours a Met residue at the putative P3′ position in its RCL. Indeed, GrM cleaves SERPINB9, however, an SI value of 59 points to a substrate rather than an inhibitor [30]. Therefore, it has been proposed that cleavage and inactivation of SERPINB9 by GrM clears the way for GrB-induced target cell killing [30]. Whether intracellular serpins other than SERPINB4 exist that inhibit human GrM activity remains an open question.
Whether SERPINB4 is a physiologic inhibitor of GrM remains to be investigated. We showed that SERPINB4 forms a typical serpin-protease SDS-stable complex with both recombinant and native human GrM (Fig. 1). Kinetic analysis of GrM-inhibition by SERPINB4 revealed a SI of 1.6 (Fig. 3A), indicating that about two-third of the SERPINB4 molecules forms a covalent complex with GrM and inhibits its activity, whereas about one-third of the SERPINB4 molecules is a substrate of GrM without trapping the protease. This corresponds with the relative intensity of the bands in Fig. 1A representing complexed and cleaved SERPINB4. The SI for SERPINB4 and GrM is in the similar range as the physiological interaction between SERPINB9 and caspase-1 (SI∼1.7) [39] and somewhat higher as compared to SERPINB9 and GrB (SI∼1.0) [40]. The inhibitory potency of SERPINB4 towards GrM seems different for a small molecular substrate (SI = 1.6; Fig. 3A) as compared to macromolecular substrates (SI>1.6; Fig. 4). The reason for this discrepancy remains unclear, but might be due to our recombinant proteins. It could be that a tiny fraction (<1%) of our recombinant GrM is not properly inhibited by SERPINB4 due to abnormal folding or glycosylation by P. pastoris, but retains its proteolytic activity. If so, this will not influence the determined SI-value significantly (Fig. 3A). However, cleavage of nucleophosmin by GrM is very efficient and occurs already at nanomolar concentrations of GrM [11], therefore a tiny fraction of non-inhibited GrM can be demonstrated by Western blot analysis (Fig. 4). The determined kinh of 1.3×104 M−1s−1 for the rate of GrM inhibition by SERPINB4 (Fig. 3B) was into a similar range as compared with other SERPINB4-protease interactions. Previously, SERPINB4 has been demonstrated to inhibit cathepsin G and chymase with kinh-values of 1.0×105 M−1s−1 and 2.8×104 M−1s−1, respectively [41]. The kinh-value for SERPINB4 and GrM is lower as compared to SERPINB9 and GrB (1.7×106 M−1s−1) [40]. However, a physiological inhibitory role has been proposed for serpins with a relative low kinh-value [32], for instance inhibition of TNF-mediated apoptosis by SERPINB4 via cathepsin G (kinh 1.0×105 M−1s−1) [26], endogenous anti-inflammatory action of SERPINB9 via inhibition of caspase 1 (kinh 7×102 M−1s−1) [39], activated protein C inhibition by Protein C inhibitor (SERPINA5) (kinh 2.5×104 M−1s−1) during anti-coagulation [42], and complement C1 and kallikrein inhibition by C1-inhibitor (SERPING1) (kinh 2.8×103−1.7×104 M−1s−1) [43]. In this context, it should also be mentioned that, like for other serpins [44], the inhibitory capacity of SERPINB4 towards GrM or other serine proteases could potentially be further enhanced by serpin cofactors, although here we excluded a role for heparin (data not shown). We provide in vitro evidence that overexpression of SERPINB4 inhibits both GrM-induced and NK cell-mediated cell death in tumor cells (Fig. 5 and 6). Whether SERPINB4 indeed is a physiological GrM-inhibitor in humans is ultimately determined by local concentrations of GrM and SERPINB4 in vivo.
In normal tissue, SERPINB4 is mainly expressed by stratified squamous epithelium of both the upper gastrointestinal tract and the female genitourinary system, and by pseudo-stratified columnar epithelium of the conducting airways [45]. Interestingly, tumors originating from these epithelial tissues also express SERPINB4, i.e. squamous cell carcinomas of the cervix [46], head and neck, and lung [45]. Moreover, elevated SERPINB4 levels in patients with squamous cell carcinomas are associated with an advanced stage of tumor progression and poor disease-free survival [47], [48]. This indicates that expression of SERPINB4 is beneficial for tumor cells. Indeed, SERPINB4 inhibits both radiation- and TNF-induced apoptosis in transfected cell lines, probably by inhibition of the p38 MAPK pathway and the proteolytic activity of endogenous cathepsin G, respectively [26], [49]. Our current findings that SERPINB4 binds to human GrM and that cellular overexpression of SERPINB4 inhibits GrM-induced cell death suggest a novel function for SERPINB4, i.e. enhancement of tumor progression through interference with the granule-exocytosis cell death pathway of cytotoxic lymphocytes. This intriguing concept has been well studied for SERPINB9-expressing tumor cells that efficiently inhibit GrB-induced cell death [17]. SERPINB9 expression in several types of tumors is associated with a poor clinical outcome of patients [19], [20]. In analogy with SERPINB9, SERPINB4 expression may constitute a novel mechanism by which human squamous cell carcinoma tumor cells evade GrM-mediated cytotoxic lymphocyte-induced cell death.
We greatly thank Selçuk Çolak, Ka Wai Lai, and Isabel Vandenberghe for excellent technical assistance.
Conceived and designed the experiments: PJAdK JAK DMW NB. Performed the experiments: PJAdK SAHdP RQ RB AFM WJH BD. Analyzed the data: PJAdK SAHdP JAK BD DMW NB. Contributed reagents/materials/analysis tools: DMW BD. Wrote the paper: PJAdK NB.
1. Barry M, Bleackley RC (2002) Cytotoxic T lymphocytes: all roads lead to death. Nat Rev Immunol 2: 401–409.
2. Smyth MJ, Kelly JM, Sutton VR, Davis JE, Browne KA, et al. (2001) Unlocking the secrets of cytotoxic granule proteins. J Leukoc Biol 70: 18–29.
3. Cullen SP, Martin SJ (2008) Mechanisms of granule-dependent killing. Cell Death Differ 15: 251–262.
4. Chowdhury D, Lieberman J (2008) Death by a thousand cuts: granzyme pathways of programmed cell death. Annu Rev Immunol 26: 389–420.
5. Bovenschen N, Kummer JA (2010) Orphan granzymes find a home. Immunol Rev 235: 117–127.
6. Bade B, Boettcher HE, Lohrmann J, Hink-Schauer C, Bratke K, et al. (2005) Differential expression of the granzymes A, K and M and perforin in human peripheral blood lymphocytes. Int Immunol 17: 1419–1428.
7. de Koning PJ, Tesselaar K, Bovenschen N, Colak S, Quadir R, et al. (2010) The cytotoxic protease granzyme M is expressed by lymphocytes of both the innate and adaptive immune system. Mol Immunol 47: 903–911.
8. Sayers TJ, Brooks AD, Ward JM, Hoshino T, Bere WE, et al. (2001) The restricted expression of granzyme M in human lymphocytes. J Immunol 166: 765–771.
9. Kelly JM, Waterhouse NJ, Cretney E, Browne KA, Ellis S, et al. (2004) Granzyme M mediates a novel form of perforin-dependent cell death. J Biol Chem 279: 22236–22242.
10. Bovenschen N, de Koning PJ, Quadir R, Broekhuizen R, Damen JM, et al. (2008) NK Cell Protease Granzyme M Targets {alpha}-Tubulin and Disorganizes the Microtubule Network. J Immunol 180: 8184–8191.
11. Cullen SP, Afonina IS, Donadini R, Luthi AU, Medema JP, et al. (2009) Nucleophosmin is cleaved and inactivated by the cytotoxic granule protease granzyme M during NK cell-mediated killing. J Biol Chem 284: 5137–5147.
12. Hu D, Liu S, Shi L, Li C, Wu L, et al. (2010) Cleavage of survivin by Granzyme M triggers degradation of the survivin-X-linked inhibitor of apoptosis protein (XIAP) complex to free caspase activity leading to cytolysis of target tumor cells. J Biol Chem 285: 18326–18335.
13. Hua G, Zhang Q, Fan Z (2007) Heat shock protein 75 (TRAP1) antagonizes reactive oxygen species generation and protects cells from granzyme M-mediated apoptosis. J Biol Chem 282: 20553–20560.
14. Lu H, Hou Q, Zhao T, Zhang H, Zhang Q, et al. (2006) Granzyme M directly cleaves inhibitor of caspase-activated DNase (CAD) to unleash CAD leading to DNA fragmentation. J Immunol 177: 1171–1178.
15. Deveraux QL, Takahashi R, Salvesen GS, Reed JC (1997) X-linked IAP is a direct inhibitor of cell-death proteases. Nature 388: 300–304.
16. Irmler M, Thome M, Hahne M, Schneider P, Hofmann K, et al. (1997) Inhibition of death receptor signals by cellular FLIP. Nature 388: 190–195.
17. Bird CH, Sutton VR, Sun J, Hirst CE, Novak A, et al. (1998) Selective regulation of apoptosis: the cytotoxic lymphocyte serpin proteinase inhibitor 9 protects against granzyme B-mediated apoptosis without perturbing the Fas cell death pathway. Mol Cell Biol 18: 6387–6398.
18. Zhang M, Park SM, Wang Y, Shah R, Liu N, et al. (2006) Serine protease inhibitor 6 protects cytotoxic T cells from self-inflicted injury by ensuring the integrity of cytotoxic granules. Immunity 24: 451–461.
19. ten Berge RL, Meijer CJ, Dukers DF, Kummer JA, Bladergroen BA, et al. (2002) Expression levels of apoptosis-related proteins predict clinical outcome in anaplastic large cell lymphoma. Blood 99: 4540–4546.
20. van Houdt IS, Oudejans JJ, van den Eertwegh AJ, Baars A, Vos W, et al. (2005) Expression of the apoptosis inhibitor protease inhibitor 9 predicts clinical outcome in vaccinated patients with stage III and IV melanoma. Clin Cancer Res 11: 6400–6407.
21. Bladergroen BA, Meijer CJ, ten Berge RL, Hack CE, Muris JJ, et al. (2002) Expression of the granzyme B inhibitor, protease inhibitor 9, by tumor cells in patients with non-Hodgkin and Hodgkin lymphoma: a novel protective mechanism for tumor cells to circumvent the immune system? Blood 99: 232–237.
22. Huntington JA, Read RJ, Carrell RW (2000) Structure of a serpin-protease complex shows inhibition by deformation. Nature 407: 923–926.
23. Kaiserman D, Bird PI (2010) Control of granzymes by serpins. Cell Death Differ 17: 586–595.
24. de Koning PJ, Kummer JA, Bovenschen N (2009) Biology of granzyme M: a serine protease with unique features. Crit Rev Immunol 29: 307–315.
25. Pegram HJ, Haynes NM, Smyth MJ, Kershaw MH, Darcy PK (2010) Characterizing the anti-tumor function of adoptively transferred NK cells in vivo. Cancer Immunol Immunother 59: 1235–1246.
26. McGettrick AF, Barnes RC, Worrall DM (2001) SCCA2 inhibits TNF-mediated apoptosis in transfected HeLa cells. The reactive centre loop sequence is essential for this function and TNF-induced cathepsin G is a candidate target. Eur J Biochem 268: 5868–5875.
27. Kummer JA, Kamp AM, van Katwijk M, Brakenhoff JP, Radosevic K, et al. (1993) Production and characterization of monoclonal antibodies raised against recombinant human granzymes A and B and showing cross reactions with the natural proteins. J Immunol Methods 163: 77–83.
28. Yagita M, Huang CL, Umehara H, Matsuo Y, Tabata R, et al. (2000) A novel natural killer cell line (KHYG-1) from a patient with aggressive natural killer cell leukemia carrying a p53 point mutation. Leukemia 14: 922–930.
29. Schechter NM, Plotnick MI (2004) Measurement of the kinetic parameters mediating protease-serpin inhibition. Methods 32: 159–168.
30. Mahrus S, Kisiel W, Craik CS (2004) Granzyme M is a regulatory protease that inactivates proteinase inhibitor 9, an endogenous inhibitor of granzyme B. J Biol Chem 279: 54275–54282.
31. Schneider SS, Schick C, Fish KE, Miller E, Pena JC, et al. (1995) A serine proteinase inhibitor locus at 18q21.3 contains a tandem duplication of the human squamous cell carcinoma antigen gene. Proc Natl Acad Sci U S A 92: 3147–3151.
32. Gettins PG (2002) Serpin structure, mechanism, and function. Chem Rev 102: 4751–4804.
33. Keijer J, Linders M, Wegman JJ, Ehrlich HJ, Mertens K, et al. (1991) On the target specificity of plasminogen activator inhibitor 1: the role of heparin, vitronectin, and the reactive site. Blood 78: 1254–1261.
34. Suck G, Branch DR, Smyth MJ, Miller RG, Vergidis J, et al. (2005) KHYG-1, a model for the study of enhanced natural killer cell cytotoxicity. Exp Hematol 33: 1160–1171.
35. Bots M, Kolfschoten IG, Bres SA, Rademaker MT, de Roo GM, et al. (2005) SPI-CI and SPI-6 cooperate in the protection from effector cell-mediated cytotoxicity. Blood 105: 1153–1161.
36. Smyth MJ, O'Connor MD, Trapani JA, Kershaw MH, Brinkworth RI (1996) A novel substrate-binding pocket interaction restricts the specificity of the human NK cell-specific serine protease, Met-ase-1. J Immunol 156: 4174–4181.
37. Barnes RC, Worrall DM (1995) Identification of a novel human serpin gene; cloning sequencing and expression of leupin. FEBS Lett 373: 61–65.
38. Silverman GA, Whisstock JC, Askew DJ, Pak SC, Luke CJ, et al. (2004) Human clade B serpins (ov-serpins) belong to a cohort of evolutionarily dispersed intracellular proteinase inhibitor clades that protect cells from promiscuous proteolysis. Cell Mol Life Sci 61: 301–325.
39. Young JL, Sukhova GK, Foster D, Kisiel W, Libby P, et al. (2000) The serpin proteinase inhibitor 9 is an endogenous inhibitor of interleukin 1beta-converting enzyme (caspase-1) activity in human vascular smooth muscle cells. J Exp Med 191: 1535–1544.
40. Sun J, Bird CH, Sutton V, McDonald L, Coughlin PB, et al. (1996) A cytosolic granzyme B inhibitor related to the viral apoptotic regulator cytokine response modifier A is present in cytotoxic lymphocytes. J Biol Chem 271: 27802–27809.
41. Schick C, Kamachi Y, Bartuski AJ, Cataltepe S, Schechter NM, et al. (1997) Squamous cell carcinoma antigen 2 is a novel serpin that inhibits the chymotrypsin-like proteinases cathepsin G and mast cell chymase. J Biol Chem 272: 1849–1855.
42. Espana F, Gruber A, Heeb MJ, Hanson SR, Harker LA, et al. (1991) In vivo and in vitro complexes of activated protein C with two inhibitors in baboons. Blood 77: 1754–1760.
43. Wachtfogel YT, Harpel PC, Edmunds LH Jr, Colman RW (1989) Formation of C1s-C1-inhibitor, kallikrein-C1-inhibitor, and plasmin-alpha 2-plasmin-inhibitor complexes during cardiopulmonary bypass. Blood 73: 468–471.
44. Patston PA, Church FC, Olson ST (2004) Serpin-ligand interactions. Methods 32: 93–109.
45. Cataltepe S, Gornstein ER, Schick C, Kamachi Y, Chatson K, et al. (2000) Co-expression of the squamous cell carcinoma antigens 1 and 2 in normal adult human tissues and squamous cell carcinomas. J Histochem Cytochem 48: 113–122.
46. Kato H, Suehiro Y, Morioka H, Torigoe T, Myoga A, et al. (1987) Heterogeneous distribution of acidic TA-4 in cervical squamous cell carcinoma: immunohistochemical demonstration with monoclonal antibodies. Jpn J Cancer Res 78: 1246–1250.
47. de Bruijn HW, Duk JM, van der Zee AG, Pras E, Willemse PH, et al. (1998) The clinical value of squamous cell carcinoma antigen in cancer of the uterine cervix. Tumour Biol 19: 505–516.
48. Kato H, Miyauchi F, Morioka H, Fujino T, Torigoe T (1979) Tumor antigen of human cervical squamous cell carcinoma: correlation of circulating levels with disease progress. Cancer 43: 585–590.
49. Murakami A, Suminami Y, Hirakawa H, Nawata S, Numa F, et al. (2001) Squamous cell carcinoma antigen suppresses radiation-induced cell death. Br J Cancer 84: 851–858.
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SKRWT - the missing link in high-end smartphone photography!
SKRWT - the missing link in high-end smartphone photography - is the most powerful keystone- and lens-correction tool out there and has been selected for the App Store's "Best Apps 2014". Discover never-before-seen editing features and enhance your smartphone photography with just a few taps. All hail symmetry!
Converging lines have always been a given in smartphone photography due to the fixed focal length of today's mobile phones – but not anymore. You can now correct horizontal and vertical perspective distortion with just one swipe. It's really that simple!
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Our custom-grid offers various options for your personal viewing pleasure both in terms of color as well as grid-size. So whatever your original photo looks like – you're in control!
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All file information at one glance: See your shots aperture, shutter, ISO and more in the built-in exif reader.
While we were at it, we've been consequently rethinking workflows. A preview before importing, immensely improving the selection process before editing.
Arrange your gallery's folders in whatever way works best - and optionally hide folders hindering your SKRWT workflow.
We've been rethinking the SKRWT experience to come up with ways to make it even easier to handle. So we overhauled the interface which now can be worked one-handed and even more intuitively - new control gestures included!
For minor mirroring purposes that don't necessarily have to go through MRRW, there's a new instant mirror filter in the main interface.
Optionally overwrite original photographs with the edited file to help prevent clustering your phone's gallery.
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text:
Q: How to change font-style on strings when brackets occur (using PHP/CSS) I'm fairly new to PHP and MySQL, but have been having a lot of fun with it in the last few months. I'm a bit stuck as to how to achieve this though, so hopefully some of you can help me out.
I have a text column in my MySQL DB that stores lines of text. I retrieve this by PHP and iterate over each line. What I want to achieve is that everything placed in these brackets; "[" and "]"  - are greyed out in another font-class (or using span-tag) by CSS when I output them. This is for displaying them in a style similar to comments. So far I've written this code to grab the string position for both brackets, but I'm stuck on how to achieve the desired effect with PHP+HTML/CSS, as the placement of the brackets differ from line to line.
if (isset ($row['setlist']))
{
    echo "Setlist:
";
    foreach(explode("\n", $row['setlist']) as $line)
    {
        $a = strpos ($line,"[");
        $b = strpos($line,"]");
        if ($a || $b > 0)
        {
            // how to use the position to change fontclass/color from position $a to $b..?
        }
        echo $line."
";}
        echo "
";}

A: You could just replace [ and ] with  and , that's much easier and should do the same.

redpajama_set_name: RedPajamaStackExchange


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We are looking for great people to join our team.
We are looking for great people to join our team, and work with us at Belfast Wealth Management. We would love to hear from you if you are looking for the next challenge in your working life, and believe you have what it takes to help us grow our boutique Financial Planning business.
Due to organic growth, as well as planned expansion in 2019, exciting opportunities exist throughout the business, as we embark on our next stage of growth. This could mean varied roles across the entire business, or you might be interested in carving out a niche that suits your own specialities.
Financial Planning- you might be a Senior Adviser, Associate, Para-Planner or Administrator.
You might be a University graduate, with a Financial Planning degree or equivalent, who is looking to the take the first, or next, step in your exciting career.
Don't be too concerned about duties or hours at this point - for the right person, we will build a role that suits you.
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Please attach a resume detailing your work experience and qualifications.
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This computer rendering shows what the 600-meter-long (2,000-foot) Ocean Cleanup system will look like floating in the North Pacific. Currents will push on the boom to form a u-shape. The system is designed to trap bits of plastic that boats can collect later.
Watch out, ocean trash! On September 8, a project to scoop plastic debris from the ocean launched its first phase from Alameda, Calif. Called Ocean Cleanup, its creators claim it that by 2040 can remove 90 percent of the plastic in the Great Pacific Garbage Patch. If true, that would be a lot of plastic.
When researchers visited the that so-called garbage patch back in 2015, they estimated that it covered some 1.6 million square kilometers (620,000 square miles). That's an area twice the size of Texas.
Boyan Slat first proposed Ocean Cleanup's system in a 2012 TED talk. At the time, he was just 18 years old. The Dutch-born inventor's system consists of many floating tubes. Known as booms, they form a long, snaking line. This barrier will collect floating plastic trash so people can come by and retrieve it. People have donated more than $30 million to fund the project, based in Delft, the Netherlands.
Not everyone, however, thinks it's a great idea. Some researchers worry the project could harm ocean life. There's also the issue of the targets' size. Ocean Cleanup is designed to capture pieces of plastic ranging from a few millimeters in diameter to tens of meters across, such as fishing nets. In fact, bits smaller than half a centimeter across — so-called microplastics — make up the majority of ocean plastic. The project has not been designed to catch such small bits.
Critics also worry the project will take attention and money away dealing with the root cause of this pollution: too much plastic waste in the first place.
Much ocean plastic is in bits too tiny for Ocean Cleanup to catch. But when team members went to the great Pacific garbage patch in 2015, they found that more than 90 percent of the plastic garbage, by mass, is in pieces larger than 5 centimeters (2 inches). Here's some of that debris.
Laurent Lebreton is Ocean Cleanup's lead oceanographer. He led a study of the Great Pacific Garbage Patch. This is an infamous collection of trash in the Pacific Ocean. The trash collects in a vast ocean swirl known as the North Pacific gyre.
A new study estimates that this patch includes about 1.8 trillion pieces of debris. Buoyant plastics, such as polyethylene (Pah-lee-ETH-eh-leen) and polypropylene (Pah-lee PRO-puh-leen), dominate. Most are pieces smaller than half a centimeter (0.2 inch) across. But by mass, most of the bits of plastic —90 percent — are pieces 5 centimeters or bigger.
Lebreton's team shared its findings March 22 in Scientific Reports.
It's those larger pieces that Ocean Cleanup has been designed to snag. Its line of floating booms 600 meters (almost 2,000 feet) long will drift on the water, driven by winds, waves and ocean currents. Currents will naturally push the line of booms into a u-shape. And that will corral plastic debris as it travels. Beneath the surface, the booms drag a 3-meter-long (10-foot) skirt. This net will let only the tiniest bits of plastic escape.
Solar-powered lights, satellite antennas and other equipment show boats where the booms are. These features should help prevent boats from colliding with the booms. They'll also help project scientists track where the floating system is. Periodically, boats will go out to the booms and ferry home much of the plastic it's holding for recycling.
A line of test booms, called "System 001," launched September 8. Ocean Cleanup has also tested parts of the system in the North Sea and off the California coast. After two weeks of testing off California, System 001 will head to the North Pacific gyre, Lebreton says. If all goes well, it should arrive within five weeks of the launch date. "We're hoping to bring the first plastic back before the end of the year," he says.
Ocean Cleanup plans to eventually have about 60 such systems. With the full fleet deployed, the group says, it should be able to remove 50 percent of the plastic in the patch within five years. By 2040, it hopes to have eliminated 90 percent — by weight.
Ocean Cleanup teams put together System 001 in Alameda, Calif., a few weeks before the launch.
Plastic is bad for the environment. But no one knows whether the boom-and-cleanup system itself might pose any harm.
Ocean Cleanup hired an outside group to investigate how the system might affect marine life. Its report, published in July, found no reasons for serious concern. But it did note one small concern: Sea turtles might be drawn to the system and then eat the trash.
There are many other issues the report didn't cover, says Kim Martini. She's a physical oceanographer at Sea-Bird Scientific. This Seattle-based company makes tools to measure ocean properties. Martini wrote about Ocean Cleanup in January 2017 for the oceanography blog Deep Sea News. In her piece, she listed some worries. For example, the booms' sweeping net could gather marine hitchhikers, from algae to barnacles and other kinds of critters. They could add drag to the system and change how well it performs, she says.
Also unknown, she says, is whether herding the plastic will cause new, tiny ecosystems to form amidst the trash. This could possibly attract even more wildlife to the area that the system might then scoop up by accident.
Another big issue, Martini says, is that there's no evidence yet that the system can actually collect much plastic. The latest California trial will be the first real test of how well the system may works.
Marcus Eriksen also doubts the system will collect as much plastic as Ocean Cleanup's team claims. He's an environmental scientist at 5 Gyres Institute. This is a nonprofit in Los Angeles, Calif. Eriksen worked with Lebreton worked on a 2014 study of ocean plastic. It estimated that there are 5 trillion bits of ocean plastic worldwide. But many of these bits quickly leave the surface, they reported in the journal PLOS ONE.
A lot of the debris washes ashore, gets shredded or sinks, Eriksen says. So many of the small particles would end up below the project's 3-meter-deep skirt. Meanwhile, new particles are always entering the ocean. That's why 5 Gyres and other conservation groups are focused on prevention, not cleanup. Persuading people to use less plastic in the first place is the best way to reduce ocean trash, Eriksen says.
The Ocean Cleanup team says its project is meant to work together with such prevention measures. For now, it's worth trying to remove whatever is already floating near the surface, they argue.
Whatever the result of their new tests, some groups are applauding Ocean Cleanup for raising awareness of the ocean's plastic trash problem.
"I love how passionate people are about the Ocean Cleanup project," says Adam Lindquist. He's director of the Healthy Harbor Initiative in Baltimore, Md. His group installed a pair of water wheels at two river mouths in his city's Inner Harbor area. Anchored to the ocean floor, the floating are have booms and skirts much like those on the Ocean Cleanup system. The two wheels are nicknamed Mr. Trash Wheel and Professor Trash Wheel. In four and a half years, they've scooped up some 900 tons of debris — ranging from contact lenses to mattresses. This was all trash kept out of the ocean.
"Any tech that's removing plastics from the ocean is a good thing," Lindquist says. But the bigger value in such programs will be their "getting people to take the problem more seriously, so that we can move toward real solutions." In other words, stopping plastic from entering the ocean in the first place.
antenna (plural: antennae) In biology: Either of a pair of long, thin sensory appendages on the heads of insects, crustaceans and some other arthropods. (in physics) Devices for picking up (receiving) electromagnetic energy.
oceanography (adj. oceanographic ) The branch of science that deals with the physical and biological properties and phenomena of the oceans. People who work in this field are known as oceanographers.
polypropylene The second most common plastic in the world. It is tough and durable. Polypropylene is used in packaging, clothing and furniture (such as plastic chairs).
Journal: L. Lebreton et al. Evidence that the Great Pacific Garbage Patch is rapidly accumulating plastic. Scientific Reports. Published online March 22, 2018. doi:10.1038/s41598-018-22939-w.
M. Eriksen et al. Plastic pollution in the world's oceans: More than 5 trillion plastic pieces weighing over 250,000 tons afloat at sea. PLOS ONE. Published online December 10, 2014. doi:10.1371/journal.pone.0111913.


text:
Criminal Minds: Evolution Season 1: How Many Episodes Are There?
Image Credit: Paramount +
Criminal Minds: Evolution has finally arrived. The long-awaited popular procedural drama revival series premiered its first two episodes on November 24th.
The show begins as the world is beginning to open back up after the pandemic. However, there's no rest for the wicked. While others were busy caring for their families and keeping their communities safe, a serial killer ring was in the making.
These killers are the biggest threat this investigative crew has ever encountered, and they'll need to stop them before anyone else gets hurts. Unfortunately, this means destroying this network, one murder at a time.
How many episodes are in Criminal Minds: Evolution?
The first season of Criminal Minds: Evolution has ten episodes. After the two-episode premiere, fans can expect one episode to be released weekly on Thursdays.
Episode 5, which debuts on December 15th, marks the midseason finale, and the show will then pause until January 12th. Once new episodes release on Paramount+, we'll be in for a ride until the finale airs on February 9th.
Here is a list of the cast members involved in criminal Minds :
Joe Mantegna as David Rossi
A.J. Cook as Jennifer Jareau
Kirsten Vangsness as Penelope Garcia
Aisha Tyler as Dr. Tara Lewis
Adam Rodriguez as Luke Alvez
Paget Brewster as Emily Prentiss
For more such updates, please refer to our website.
In this article:Criminal Minds, Criminal Minds: Evolution, Criminal Minds: Evolution season 1, trillmag


text:
Kendrick Lamar makes a thrilling return to…
ReviewReview An assessment or critique of a service, product, or creative endeavor such as art, literature or a performance.
Kendrick Lamar makes a thrilling return to Anaheim's Honda Center with The Big Steppers Tour
The concert will be followed by one night at Toyota Arena in Ontario on Sunday, and then four more Wednesday through Saturday at Crypto.com Arena in Los Angeles.
Kendrick Lamar played Honda Center in Anaheim on Wednesday, Sept. 7, 2022, but did not allow photographers to shoot the show. He is seen here performing at the Glastonbury Festival in Worthy Farm, Somerset, England, Monday, June 27, 2022. (AP Photo/Scott Garfitt)
By Peter Larsen | plarsen@scng.com | Orange County Register
PUBLISHED: September 8, 2022 at 2:30 p.m. | UPDATED: September 8, 2022 at 7:32 p.m.
Kendrick Lamar had been away a long, long time.
One thousand eight-hundred and 55 days between the release of his album "Mr. Morale & the Big Steppers" and its Pulitzer Prize-winning predecessor "DAMN."
Lamar's last live shows in Orange or Los Angeles counties were in 2018. The concert Wednesday, Sept. 7, is the first of six in the region, followed by Sunday, Sept. 11 at Toyota Arena in Ontario, then Wednesday-Saturday, Sept. 14-17, at Crypto.com Arena in Los Angeles.
Kendrick Lamar with a lookalike ventriloquist puppet at the start of his concert at Honda Center in Anaheim on Wednesday, Sept. 7, 2022. (Photo by Greg Noire)
It was, as our narrator for the show, Helen Mirren – yes, really – proclaimed Wednesday, time for Kendrick to get out of the house again.
"Mr. Morale, this is Helen Mirren," our favorite dame declared in the first of her periodic recorded announcements throughout the show. "I'll be guiding you through this performance. You've been in your comfort zone for over 1,855 days. You have to get out eventually."
And Lamar did just that spectacularly in a show that packed 28 songs into an hour and 45 minutes.
It was a show that at times felt more like theater or performance art than a hip-hop concert, as the artist from Compton explored and exposed his interior life and all its messy, questioning emotions, and proved that even after 1,855 days of silence there are still very few rappers who can approach his genius.
A giant cream-curtained cube surrounded the main stage as a string section started to play just after 9 p.m. Eleven dancers, seven men in dark suits, four women in white, marched in loose-limbed syncopation toward it down a long ramp that split the crowd on the floor.
Curtains up, the stage revealed Lamar at a piano, playing the opening chords of "United In Grief," and then jumping into the rapid-fire rap of its opening verses.
Oh, and look there, it's a ventriloquist puppet in the rapper's own image that Lamar carried along with him through the rest of that song and "N95" that followed.
Ten minutes in and it's clear that this is not going to be your typical concert.
"Mr. Morale & the Big Steppers," Lamar's fifth album, arrived in May as a fascinating, raw look at what's been on his mind since last we heard new music from him.
On the double album, he explores issues of trauma and grief, double standards and expectations, his triumphs but maybe more often his failings.
"I went and got me a therapist," he raps on "United In Grief." And as Helen Mirren popped up in recorded moments interspersed throughout the concert, it became clear that she wasn't playing so much the narrator as she was Lamar's therapist.
"Mr. Morale, you once again let your ego get the best of you," Mirren tells him at the conclusion of "Rich Spirit," one of 13 songs pulled from the new record.
"First, you have to get past yourself," she advises later, before Lamar, during the new song "Count Me Out," raps, "Wipe my ego, dodge my pride," while the screens on stage display the words, "A mask won't hide who you are inside …".
It was a fascinating glimpse into the mind of a restlessly creative artist: Never indulgent, ever entertaining.
Highlights early in the show included the arrival of several of the songs from 2017's "DAMN." including "ELEMENT." sung from the center of the ramp, his silhouette looming large, and "HUMBLE." done on the main stage with the besuited dancers loping around him in intricate, precise choreography.
A handful of older songs drew huge cheers from fans from opening notes, though some, such as "Swimming Pool (Drank)" and "m.A.A.d. city" seemed truncated with Lamar letting the crowd pick up large chunks of the vocals. "King Kunta" was not trimmed, and thankfully so – it remains one of his most purely funky hits.
In the latter half of the show, Lamar worked further down the ramp, the production creating unexpected visual effects to enhance the songs and performance. For "Money Trees," a large rectangular light box descended from the rafters to create a claustrophobic fluorescent ceiling hovering above him.
For "Alright," one of the highlights of the second half of the show, a plastic-walled cube lowered over him and four dancers dressed in gleaming white hazmat suits – Mirren ordered a COVID test be taken – and he stayed inside it as it was elevated above the stage and filled with smoke during "Mirror" and "Silent Hill."
Lamar's cousin Baby Keem and Tanna Leone opened the show Wednesday, with Baby Keem earning a response far more rapturous and raucous than just about any opening act we've seen in years. He was a big draw at Coachella in April and seems poised to play his own arenas in the not-too-distant future.
Lamar brought Baby Keem back to play a trio of Keem's songs near the end of the night, and again, the crowd went wild the minute they launched into "vent," "range brothers" and "family ties." (Leone joined Lamar for his penultimate song, "Mr. Morale."
That song was sandwiched between "Crown" – "I can't please everybody," Lamar rapped on the chorus – and the final number "Savior" – "Kendrick made you think about it/But he is not your savior" that one begins.
It's clear he's been thinking about a lot and asking a lot of questions. In these concerts, if you're listening, you may well end up doing the same on the long ride home.
Parody metal band Mac Sabbath serves up delicious fun at The Observatory
Young the Giant will headline a hometown show at FivePoint Amphitheatre
Field Trip Fest returns with Jamestown Revival, Robert Jon & The Wreck and more
Peter Larsen | Reporter
Peter Larsen has been the Pop Culture Reporter for the Orange County Register since 2004, finally achieving the neat trick of getting paid to report and write about the stuff he's obsessed about pretty much all his life. He regularly covers the Oscars and the Emmys, goes to Comic-Con and Coachella, reviews pop music, and conducts interviews with authors and actors, musicians and directors, a little of this and a whole lot of that. He grew up, in order, in California, Arkansas, Kentucky and Oregon. Graduated from Lewis and Clark College in Portland, Ore. with degrees in English and Communications. Earned a master's degree at the Medill School of Journalism at Northwestern University. Earned his first newspaper paycheck at the Belleville (Ill.) News-Democrat, fled the Midwest for Los Angeles Daily News and finally ended up at the Orange County Register. He's taught one or two classes a semester in the journalism and mass communications department at Cal State Long Beach since 2006. Somehow managed to get a lovely lady to marry him, and with her have two daughters. And a dog named Buddy. Never forget the dog.
plarsen@scng.com
Follow Peter Larsen @PeterLarsenBSF
More in Music + Concerts
Grammys 2023: How to watch and who's performing at the ceremony


Sometimes, it feels like Drannik is a Mary Sue of sorts, and I want to keep him away from that image as much as possible. He may be incredibly smart, but he's still only a kid, and he doesn't always have the solution to a problem at hand.
As for this story: will Jameel lose his house? I'm keeping that up in the air for now. Keeps people reading.


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text:
Q: Can't find form element I have used jquery to load some html from another page and added the content to a div called 'console'. This happens when I press a link:
$ ('# Console'). Load (this.href + '# container');
I try then to retrieve the form which is part of the added html.
I use $('#formname') but I can not get the form element.
Why? What should I do?

A: Check your html you are putting in does the form also have an id attribute? As # is used in jQuery as the id selector.
$("[name=formname]") // if you need to use the name

Selectors: http://api.jquery.com/category/selectors/

A: The .load() method is lower case, like this:
$('#Console').load(this.href + ' #container');

Also make sure you're looking for it after it loads, like this:
$('#Console').load(this.href + ' #container', function() {
  var form = $('#formid');
});

A: Make sure you are trying to retrieve the form on complete of the ajax load. Ajax is happening asynchronously remember.
should look like this:
$('#Console').load(this.href + ' #container', function(){
    $('#formname'); // get form here
});

also check you syntax in the load url string. Need to have a space before the '#container'


text:
Home › News › Top News
Herman Cain Believes 'Fake News' Has 'Brainwashed' Black Folks To Hate Trump [Video]
NY Magee, EURWeb.com
Former presidential candidate Herman Cain believes Black folks have been "brainwashed" by the media to hate the president.
Cain, Donald Trump's former nominee to serve on the Federal Reserve Board, was asked by TMZ why more Black Americans hold less than favorable views of Trump despite his progress on criminal justice reform, for instance.
"They're getting brainwashed according to the news that they watch," Cain said. "It's been statistically shown that certain stations, certain news outlets, they simply are not telling the entire truth. And in some cases, people are being brainwashed."
Herman Cain Says Black People Are Brainwashed to Hate President Trump https://t.co/dmJugPdV24
— TMZ (@TMZ) May 14, 2019
Twitter user Aisha Na'Sha replied under the comments of the TMZ story with: "Definitely not true. My earliest memory of Donald Trump was him doing something disrespectful. So from jump my opinion on him has always been a little off. I don't hate anyone."
Cain says Trump should be acknowledged for the work he's doing behind the scenes for the culture, like pushing forward criminal justice reform and lowering unemployment rates. He believes these accomplishments are overlooked due to "fake news."
Meanwhile, as noted by The Hill, Trump announced Cain's withdrawal from consideration to serve on the Federal Reserve's board of governors in a tweet days after the pizza tycoon said on the Fox Business Network that he wouldn't "run away from criticism" and that withdrawing was "not in [his] nature."
"My friend Herman Cain, a truly wonderful man, has asked me not to nominate him for a seat on the Federal Reserve Board. I will respect his wishes. Herman is a great American who truly loves our Country!" Trump tweeted last month.
10 Black Trump Supporters
black people and trump	, Donald Trump	, Herman Cain