The world is your oyster, but that can make it hard to narrow down a destination when it comes to travel. Whether you're looking for your next big trip overseas or just need a quick weekend getaway nearby, there are plenty of resources for you to find out the best places to visit. You should start by reading through travel sites, a travel guide or magazine, a book or even a travel guide blog to get the inside scoop on the topic of travel. It's a good idea to pick a resource with a pictures gallery so you can get a good sense of whether each place is right for you or get ideas about the best things to do when you arrive.
Traveling expenses can really add up when it comes to preparing for and going on a journey. Luckily, there are tools available throughout the whole process to ensure that you don't overspend! We highly recommend starting with an app to manage your travel budget, so you can properly save for your big vacation and track all of your expenses along the way. It's also a good idea to use apps to book cheap flights and compare hotel options or accommodations to be sure you're getting the best deals out there and have more money to spend on things like experiences, food, and souvenirs. You might even want to look into vacation packages or bundles to get even better discounts.
World travel can really be the journey of a lifetime, but a little more planning is required to be sure your trip goes off without a hitch. Every world traveler should have a good trip planning app to make a checklist of everything they need to do before their flight from travel guide research to acquiring a passport and even packing the right items. Also when you're traveling around the world, you'll need to be able to find top restaurants for food and drink and other fun attractions so you really get the full experience of a new country and culture. You'll be thankful that you properly prepared so that you have a stress-free and amazing time.


Remove Moss or Replace Roof? Your Choice.
The cousins to home renovations are home repair and maintenance. A person remodels a house because it's no longer stylistically suitable; because it's not meeting functional needs; or because major repairs need to be made, and it turns out easier in the end just to completely remodel the thing.
No one has ever replaced an otherwise functional roof simply because it doesn't meet stylistic needs.
In most cases, roofs get replaced because they have reached the end of their natural lifespan or they have not been properly maintained.
If you maintain your roof well enough, you probably will not be replacing it. Have I driven the point home enough yet? I hope so, because now we're going to kill some moss!
Moss develops in perennially shady areas in areas that tend to be cool and damp. You can live in a cool, damp area, but sections of the roof that get a couple of hours of sun per day may not develop moss. It's that third factor–lack of sun–that promotes moss growth.
As moss thickens, it will get under the shingles, acting like a jack and raising and pushing them upward. It's bad enough with asphalt or composite shingles, but if you have wood shingles it's disastrous. Wood's porous surface is prime real estate for moss growth. Once moss has adhered to wood shingles, it's tougher to get off than the relatively smoother planes of composite or asphalt shingles.


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In 1982 in Digoin, a small town in Burgundy, France Passion Plaisance was born through the initiative of a handful of passionate people. They were among the first to offer the possibility to discover their region from aboard a houseboat.
More than just a travel agency, FPP is also a constructor of canal boats. Currently their famous Tarpon (a classic among canal boats) sails on the rivers and canals all over Europe.
FPP TRAVEL is an agency specialising in river and canal tourism, created by a small group of enthusiasts eager to share the many joys of cruising on licence free boats, and discovering the hidden treasures of different regions.
Handy: accessible to people with a disability, labialized "Tourism and Handicap"
Experience : Véronique has worked on Canalous bases and for 22 years within the sales department of FPP Travel.
My experience of over 20 years in the Canalous Group is a guarantee of my knowledge of boating. I know tell you about all our destinations, particularly on the Roanne canal, Camargue, Herault, Aude, Lot and Burgundy.
Have a nice cruise with France Passion Plaisance!


Today marks a month of being engaged and how fitting that today also marks the start of the proposal story!!! Enjoy!
Sometimes Jeffrey and I will reminisce about time past. We both had been praying for friends. I had been praying for some solid guy friends, Jeffrey had been praying for some friends in general. So when we started hanging out both of our prayers were answered! It has been so cool to see God's hand in our relationship. That doesn't mean it's been perfect. We've hurt each other in so many ways and have wished we could turn back time and do 'that moment' again, but so is life. I hope that every time we've moved forward we have learned from our mistakes and do better in the next situation.
I was about to be off to a week of camp that came up short notice. I had asked Jeffrey if there was any reasons he felt I shouldn't go and it seemed like something was bothering him, but he said he was leaving it in God's hands. At the time I was slightly confused by that statement but later I found out that he had a day picked out that he wanted to propose but I was now going to be gone that day. I was slightly naive... but it was hard not to notice that Jeffrey had been acting a little weird of late. You might say I was becoming slightly suspicious. We had talked a lot about engagement and marriage and Jeffrey had a timeline for himself; get engaged by fall 2017 or don't date at all. It seems harsh but I felt the same… if we had been dating for two years and had no plans of moving forward to marriage then what was the point of "dating for marriage" and being intentional?! So with that knowledge I knew it was coming. Every time I saw Jeffrey there was always the thought that maybe today was the day.
Jeffrey and I both love photography and have gotten together often for a photoshoot so on August 18, 2017 when he texted me asking if he could come after work the next day for a photoshoot there was nothing unusual about it. I also cut his hair regularly and he asked for a haircut before the shoot… now that was unusual! I'm typically after him to get it done! Now he asked?! And there was no "apparent" special occasion… I texted my friend about it because it was odd, she brushed it off as "he wants to look top notch for the shoot! Regan there's nothing really weird about it.", little did I know that she knew exactly what was going on, and had known for over a month. Jeffrey then texted me asking if I thought Rachel would be willing to take some pictures of us. I told him to ask her. The morning of August 19, 2017 Rachel came up and grumbled "Jeffrey asked me to take pictures of you and him.". Since she seemed so put out I suggested that our youngest sister could take them, to which Rachel promptly replied that she would do it. Jeffrey had told Rachel that he was proposing and wanted it photographed… she knew but was trying to act put out in front of me. The day pasted normally and we finally came to supper time! Jeffrey came for supper that evening after work and then I cut his hair. We got changed into some dressier clothes and went outside to take the pictures, Jeffrey weirdly brought his backpack with him… no comment from me though…little did I know he was hiding a ring box in there.


The third value of the animation-fill-mode property is both.
It combines the effects of forwards and backwards. That is, before the animation starts, the element gets the state of the first keyframe. When the animation is over, the final state of the animation is preserved.
The animation-fill-mode: both action affects both repeated and alternating animation.
Let's conduct our final experiment!
and then an alternating direction of animation.


text:
Topographic maps > United States of America > United States Virgin Islands
United States Virgin Islands topographic maps
United States of America > United States Virgin Islands > Cruz Bay
Cruz Bay, Saint Thomas - Saint John District, United States Virgin Islands, 000830, United States of America (18.31292 -64.78665)
Green Cay
United States of America > United States Virgin Islands
Green Cay, Saint Thomas - Saint John District, United States Virgin Islands, United States of America (18.30992 -64.90812)
Savana Island
Savana Island, Saint Thomas - Saint John District, United States Virgin Islands, United States of America (18.34108 -65.07824)
Coordinates: 18.33501 -65.08534 18.34714 -65.07383 - Minimum elevation: 0 ft - Maximum elevation: 352 ft - Average elevation: 3 ft
United States of America > United States Virgin Islands > Charlotte Amalie
Charlotte Amalie, Saint Thomas - Saint John District, United States Virgin Islands, 00804, United States of America (18.34114 -64.93279)
Frederiksted
United States of America > United States Virgin Islands > Frederiksted
Frederiksted, Saint Croix District, United States Virgin Islands, 00870, United States of America (17.71185 -64.88136)
United States of America > United States Virgin Islands > Christiansted
Christiansted, Saint Croix District, United States Virgin Islands, United States of America (17.74395 -64.70798)
Frederiksted, Christiansted, Charlotte Amalie, Cruz Bay.

redpajama_set_name: RedPajamaCommonCrawl


Defense attorney Charles Swift spent plenty of time cross-examining witnesses from the State in the Noor Salman trial. She is accused of aiding and abetting her husband Omar Mateen in the mass murder of 49 Orlando citizens at Pulse. He would stop and linger, taking time to ask questions that might raise doubt in the jury's mind. The jury consists of 12 woman and 6 men. 12 are on the actual jury and 6 are alternates. None of them has been informed if they are the alternates in order to guarantee that they all pay close attention to the details of the case.
FBI evidence collection expert Lauren Regucci took the stand to describe the steps taken to document the evidence collected from the van that Omar Mateen parked near the Pulse Nightclub. Agents are trained to draw maps that help clarify where items were found along with photos, where items were found. The vehicle was parked under a carport near a boat. The windows had been blown out by a bomb-seeking robot. A 38 revolver pistol was found near the vehicle along with items known as speed loaders. Speed loaders allow all the bullets to be loaded at once saving time when you want to kill fast.
Also found near the vehicle were a green rifle bag and a long gun rifle case. A child's stroller was also on the ground on its side. It was a strange combination of the domestic mixed with the murderous. Lauren held up a pistol box along with each item found so that the jury could see. The SIG Sauer MCX assault rifle was found next to the gunman's dead body inside the club bathroom, so it was not among the items found outside.
Tampa FBI Special Agent Lynn Billings was in charge of collecting evidence inside the club. She showed the jury a photo of Omar Mateen's cell phone that was found submerged in a pool of blood. Each item in the club was assigned a yellow marker and the distances between items was noted. She held up the the rifle purchased on June 4, 2016 by Omar Mateen and verified that the serial number on the gun matched the serial number on the receipt from Saint Lucie Gun Sales. The gun was submitted into evidence. There was a knife found next to Omar Mateen's body along with the Glock handgun that he had also purchased in the weeks leading up the the massacre. The gun holster was found on the gunman's body. Thirty-seven other bodies were found in the club. "There were cellphones ringing," she said. "People were constantly calling cellphones that were scattered throughout the club."
The FBI spent nine days collecting evidence. They measured distances between objects and figured out the shooting trajectories of shots whenever possible. They then proceeded to tear out the drywall to collect any other bullets that had penetrated the walls. Tape on the walls marked places where bullets had entered. Furniture as well was dissected for evidence collection. Besides collecting near Omar's rental van and inside the club, a map was also set up that was used to make sure any evidence in the blocks around the club was collected. Every imaginable area was searched before Orange Avenue could be once again opened up to traffic.


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A group of eight legislators younger than 45 banded together Tuesday to launch the Kansas Future Caucus, a bipartisan coalition aimed at giving young people a voice in government.Rep. Stephanie Clayton, an Overland Park Republican, and Rep. Brandon Whipple, a Wichita Democrat, will serve as co-chairs of the newly-formed group announced at a Statehouse press conference. Clayton said there are about 25 legislators who are younger than 45.
A group of eight legislators younger than 45 banded together Tuesday to launch the Kansas Future Caucus, a bipartisan coalition aimed at giving young people a voice in government. Rep. Stephanie Clayton, an Overland Park Republican, and Rep. Brandon Whipple, a Wichita Democrat, will serve as co-chairs of the newly-formed group announced at a Statehouse press conference.
Overland Park Rep. Stephanie Clayton, a Republican, and Wichita Rep. Brandon Whipple, a Democrat, today will announce the formation of a new group of legislators that will focus on issues affecting Kansans under the age of 45.
Today, Representatives Stephanie Clayton (R-19) and Brandon Whipple (D-96) announced the creation of the bipartisan Kansas State Future Caucus, a brand-new caucus comprised of state legislators under age 45. With the launch, Kansas becomes the 21st state to join Millennial Action Project's national State Future Caucus Network.
Roughly 140 Kansan's gathered at Olathe City Hall on January 18 to hear from lawmakers, journalists and legal experts about ways to fight secrecy in state government. Rep. Stephanie Clayton, Kansas Future Caucus Co-Chair, participated on the panel.
Interested in helping us grow the Kansas Future Caucus?


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A Monstrous Truth: Even after the departure of a madman the political madness remains
Posted by John Pavlovitz | Feb 9, 2021
We fooled ourselves, good people. For the last four years we told one another a necessary story.
Because we wanted to believe the best about humanity, many of us clung to a false narrative of otherwise decent people hopelessly swept up in the fervor of the moment: good human beings intoxicated in the bloody water of a feeding frenzy power-grab, eyes rolled back and unable to do anything but consume.
We wanted to believe it was a temporary condition.
We imagined that if he were gone, that once this messianic monster was removed and severed from them, that those around him would begin to come to their senses and the thick haze of their sycophantic adoration would lift and they would slowly but certainly revert to their better natures.
But now we are realizing the most painful truth of all the lessons of these years: this is simply who they are.
It turns out that the recently departed and disgraced madman was not the manipulative puppet master animating them into acts of viciousness and cruelty they'd otherwise refuse to consent to, they were the ones pulling the strings all along. What we're seeing now is irrefutable evidence that these people were not altered by the presence of some singularly evil man — but fully embodied in him.
In the first few hours following the January 6th terrorist attack, a few of them may have been briefly sobered by the loss of life or momentarily afflicted with a sudden twinge of humanity, but that proved only a fleeting moral annoyance they've since cast aside. They would not allow decency to fully overthrow their hearts.
They have newly declared their allegiance to him once more, defended the violence he instigated, loudly castigated those demanding consequences, doubled down on their social media assaults, and rushed headlong to fill the space he's left.
And today, the danger is as great as it ever was.
This vile thing is still alive and well: the slithering, lurking, ravenous, toxic creature is simply reconfiguring and reconstituting itself in order to emerge in a less obvious form, one that will not as easily be dealt with—which makes this moment more urgent than any that came before it.
Evil adapts to keep itself alive, and we need to adapt to kill it. We cannot be lulled into complacency by the euphoria of the moment, sedated into inaction by the return of rational, compassionate leadership in the Presidency or distracted by the cheap high of a political win.
We who have kept our eyes open for the past four years cannot afford to fall asleep now. We need to be more diligent than we have ever been, relentless in our collective efforts for justice, steadfast in exposing and condemning this inhumanity and not permitting it to go unabated.
If we allow this thing to remain alive and to gain strength, we will soon find our Republic mortally wounded, and these present victories will have simply postponed our inevitable demise.
But the beauty of the moment is that we know this, that we see with absolute clarity the urgency of this day, that we are capable of transcending political and religious affiliations and assembling around our shared humanity in order to defend it.
We will not fall asleep or be tempered by phony calls for unity or forget what our eyes have seen or allow ourselves to be shamed into silence.
And we will remember that we are not simply terrified potential victims, cowering in a corner just waiting to be devoured, we are fearless warriors for the common good who will not allow malevolence to have the last, loudest word.
Yes, evil dies hard, but goodness will not relent.
Brеndаn Smіаlоwskі
The original version of this Op Ed was published on johnpavlovitz.com
John Pavlovitz launched an online ministry to help connect people who want community, encouragement, and to grow spiritually. Individuals who want to support his work can sponsor his mission on Patreon, and help the very real pastoral missionary expand its impact in the world.
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John Pavlovitz is a 20-year ministry veteran trying to figure out how to love people well and to live-out the red letters of Jesus, both online in this global community.
Rapid Transit plan outlined at public forum
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Peaceful protesters in Kenosha march with Jacob Blake's family demanding an end to police brutality


text:
Heteractis magnifica ook wel Ritteri anemoon (of: Radianthus ritteri) genoemd is een zeeanemoon van de koraalriffen uit de Grote Oceaan en Indische Oceaan die wel 1 meter groot kan worden. De anemoon heeft een voorkeur voor de meer ondiepe riffen met een sterke stroming. Hij leeft in symbiose met diverse soorten anemoonvissen waarmee zijn lila of gelige kleur kan contrasteren. De tweeband anemoonvis (Amphiprion bicinctus) en de rifbaars Dascyllus trimaculatus gebruiken deze anemoon vaak als gastheer.

Zeeanemonen

redpajama_set_name: RedPajamaWikipedia


Multidrug resistance (MDR) is a major obstacle to the treatment of gastric cancer (GC). Using a phage display approach, we previously obtained the peptide GMBP1, which specifically binds to the surface of MDR gastric cancer cells and is subsequently internalized. Furthermore, GMBP1 was shown to have the potential to reverse the MDR phenotype of gastric cancer cells, and GRP78 was identified as the receptor for this peptide. The present study aimed to investigate the mechanism of peptide GMBP1 and its receptor GRP78 in modulating gastric cancer MDR.
Fluorescence-activated cell sorting (FACS) and immunofluorescence staining were used to investigate the subcellular location and mechanism of GMBP1 internalization. iTRAQ was used to identify the MDR-associated downstream targets of GMBP1. Differentially expressed proteins were identified in GMBP1-treated compared to untreated SGC7901/ADR and SGC7901/VCR cells. GO and KEGG pathway analyses of the differentially expressed proteins revealed the interconnection of these proteins, the majority of which are involved in MDR. Two differentially expressed proteins were selected and validated by western blotting.
GMBP1 and its receptor GRP78 were found to be localized in the cytoplasm of GC cells, and GRP78 can mediate the internalization of GMBP1 into MDR cells through the transferrin-related pathway. In total, 3,752 and 3,749 proteins were affected in GMBP1-treated SGC7901/ADR and SGC7901/VCR cells, respectively, involving 38 and 79 KEGG pathways. Two differentially expressed proteins, CTBP2 and EIF4E, were selected and validated by western blotting.
This study explored the role and downstream mechanism of GMBP1 in GC MDR, providing insight into the role of endoplasmic reticulum stress protein GRP78 in the MDR of cancer cells.
Gastric cancer (GC) remains the fourth most common malignancy and the second leading cause of cancer-related death worldwide . Although surgery is effective for most patients, chemotherapy remains the primary treatment for advanced gastric cancer ; nonetheless, therapies often fail due to the multidrug resistance (MDR) exhibited by some cancer cells. MDR is a phenomenon in which cancer cells that are exposed to one anti-cancer drug become resistant to several other chemotherapy drugs that are structurally and functionally different from the initial drug [3,4]. MDR is a multifactor event in which several mechanisms act simultaneously, including increased drug efflux, DNA repair activity, and altered survival and apoptotic signaling pathways [5-7]. Although there have been many pathogenesis studies on tumor MDR, the mechanisms of MDR are intricate and have not yet been fully elucidated . Moreover, there is an urgent need to find novel approaches to reverse MDR in GC.
Short peptides with rapid blood clearance, high tissue penetration and diffusion, non-immunogenicity and a high affinity for target tumor cells have attracted great interest in recent years [9-11]. In a previous study using a phage display approach, we analyzed a peptide, GMBP1, that was specifically bound to the surface of MDR gastric cancer cells and that had the potential to be internalized into these cells and reverse the gastric MDR phenotype. GRP78 was later identified as a receptor for this peptide . Importantly, exploring novel agents that can reverse MDR in GC is necessary for the improvement of chemotherapy in GC patients.
Proteomics is used as a powerful tool to accurately monitor and quantitatively detect changes in protein expression in response to drug treatment, and this approach has been widely used to investigate the mechanisms of action of chemicals on cancer cells [13-15]. Some technologies have been widely used in proteomics, including 2DE, SILAC, 2D-DIGE, and iTRAQ [16-20]. 2DE is an important proteomic technique and is widely used in comparative studies of protein expression levels. However, this technique has several disadvantages, including poor reproducibility between gels, low sensitivity in the detection of proteins in low concentrations and hydrophobic membrane proteins, limited sample capacity and a low linear range in visualization procedures [21,22]. Furthermore, only a limited number of proteins have been identified using the existing techniques. iTRAQ-based analysis, a technique that has been developed to quantitatively investigate changes in protein abundance in various biological samples with high accuracy and reproducibility [23,24], enables the differential labeling of peptides from distinct proteomes. In addition, the use of iTRAQ reagents with four to eight different tags allows for multiplexing ability . High-throughout techniques can be used to screen MDR-related proteins and to study the mechanisms of gastric cancer drug resistance, and proteomics-based iTRAQ is an excellent choice for studying MDR mechanisms. Indeed, this approach has been successfully employed to identify differentially expressed proteins in gastric cancer .
Adriamycin and vincristine have been used to treat various cancers, and these drugs are accepted worldwide as first-line anti-cancer drugs for GC chemotherapy. However, their use remains limited because of the rapid development of MDR; thus, it is necessary to explore the mechanisms underlying this resistance. To further characterize the mechanisms of MDR, adriamycin-resistant SGC7901/ADR cells and vincristine-resistant SGC7901/VCR cells, which have been widely employed as cell culture models to investigate the mechanism underlying MDR in gastric cancer, were used in this study. These cell lines were derived from the human gastric cancer cell line SGC7901 by stepwise selection in vitro using adriamycin and vincristine and developed cross-resistance to other anticancer drugs, including cisplatin, adriamycin, etoposide, mitomycin C, and 5-fluorouracil (5-FU) . Methods including FACS and immunofluorescence staining were used in this study to investigate the mechanism underlying the internalization of GMBP1. In addition, an iTRAQ-based proteomic approach coupled with bioinformatics, including GO and KEGG analyses, were also applied. Our work elucidates the molecular mechanism of GMBP1-induced reversal of MDR in GC, and the results presented here will undoubtedly provide important clues to the mechanisms of MDR in gastric cancer.
Human MDR gastric adenocarcinoma adriamycin-resistant SGC7901/ADR and vincristine-resistant SGC7901/VCR cell lines were derived in our laboratory from the human gastric cancer cell line SGC7901 by stepwise selection in vitro using adriamycin and vincristine, respectively. The cells were cultured in RPMI-1640 medium (Thermo Scientific Hyclone, Beijing, China) containing 10% fetal bovine serum, 100 μg/ml streptomycin and 100 U/ml penicillin and incubated at 37°C with 5% CO2 in a humidified incubator. To maintain the MDR phenotype, vincristine (final concentration, 1 μg/ml) was added to the culture medium of the SGC7901/VCR cells, and adriamycin (final concentration, 0.5 μg/ml) was added to the culture medium of the SGC7901/ADR cells. Adriamycin (ADR) and vincristine (VCR) were dissolved in normal saline at the indicated concentrations.
For knockdown of GRP78, GC cells were transfected with a small interfering RNA (siRNA) targeting GRP78: sense 5′-GGAGCGCAUUGAUACUAGATT-3′ and antisense 5′-UCUAGUAUCAAUGCGCUCCTT-3′ . siRNA targeting green fluorescent protein (GFP) was purchased from GenePharma (Shanghai, China) and served as a negative control. Both siRNAs were used at a final concentration of 80 nmol/l. The cells were transfected in six-well plates according to the manufacturer's instructions. Ten microliters of each siRNA was used with 5 μl of Lipofectamine 2000 per well. The transfected cells were monitored for GFP under a fluorescence microscope.
Cells were cultured on cover slips and fixed with acetone at 4°C for 30 min, blocked with 10% normal rabbit serum, and incubated with a goat anti-human GRP78 antibody (1:500; Abcam, USA) overnight at 4°C. Subsequently, the cells were incubated with a secondary FITC-conjugated anti-goat antibody (1:1,000; Invitrogen, CA, USA) for 1 h at 37°C. A drop of Prolong Gold anti-fade reagent with DAPI (Invitrogen, CA, USA) was added before the cell images were acquired using a FLUOVIEW FV1000 laser scanning confocal microscope (Olympus, Tokyo, Japan). PBS and control siRNA were used as a negative control.
Cells were cultured in serum-free RPMI-1640 medium. After 24 h, the cells were trypsinized, centrifuged at 1,000 rpm for 5 min, harvested and washed with ice-cold PBS twice. The expression level of GMBP1-GRP78 was measured by staining the cells with FITC-conjugated GMBP1 in PBS containing 0.05% bovine serum albumin on ice. FITC-GMBP1 (200 μg/ml) was incubated with the cells in growth medium for 1 h at 37°C, and the cells were then washed twice with ice-cold PBS. After removing unbound FITC-GMBP1 by washing the cells extensively in PBS, the surface immunofluorescence of viable cells was measured using a flow cytometer. FITC-URP was used as a negative control.
Cells were seeded on cover slips at a density of 106 cells/ml; experiments were conducted at 24–72 h post-seeding. The multidrug-resistant gastric cells SGC7901/ADR and SGC7901/VCR with GMBP1 were doubly labeled as follows. Briefly, the cells were serum-starved for 2 h in RPMI-1640 medium. The cells were first incubated with FITC-GMBP1 in growth medium at 200 μg/ml for 1 h at 37°C in the dark and then washed twice with ice-cold PBS. The cells were then incubated with Alexa Fluor 594-transferrin (25 μg/ml) at 4°C for 3 h in the dark to stop receptor-mediated endocytosis ; the cells were then incubated at 37°C for 30 min to initiate the uptake of FITC-GMBP1, after which the cells were washed twice with ice-cold PBS. The cell nuclei were stained using 4, 6-diamidino-2-phenylindole (DAPI). Cell images were acquired using a FLUOVIEW FV1000 laser scanning confocal microscope (Olympus, Tokyo, Japan).
The treated and untreated SGC7901/ADR and SGC7901/VCR cells were harvested and lysed in lysis buffer and centrifuged at 15,000 rpm for 30 min at 4°C. The supernatants were collected, and the total protein concentration was determined using a Bradford protein assay kit. For each sample, 100 μg of protein was precipitated by adding six volumes of cold acetone and incubating at −20°C for 4 h. The precipitated protein was dissolved in solution buffer and denatured, and the cysteines were then blocked according to the manufacturer's instructions (Applied Biosystems). Each sample was digested with 20 μl of 0.25 μg/μl trypsin (Promega) solution at 37°C overnight. iTRAQ labels 113 and 118 were used to separately label the control samples SGC7901/ADR and SGC7901/VCR, respectively, and the labels 115 and 119 were used to label the corresponding GMBP1-treated samples. The labeled samples were pooled before further analysis.
To reduce sample complexity during the LC-MS/MS analysis, the pooled samples were diluted 10-fold with HPRP buffer A (10 mM KH2PO4 in 25% acetonitrile at pH 3.0) and separated using a 2.1 × 200 mm polysulfoethyl A HPRP column (Poly LC, Columbia, MD, USA). The column was eluted with a gradient of 0–25% HPRP buffer B (10 Mm KH2PO4 at pH 3.0 in 25% acetonitrile containing 350 mM KCl) over 30 min followed by a gradient of 25-100% HPRP buffer B over 40 min. The fractions were collected at 1-min intervals. These HPRP fractions were lyophilized in a vacuum concentrator and subjected to C18 clean-up using a C18 Discovery DSC-18 SPE column (Thermo). The cleaned fractions were then lyophilized again and stored at −20°C until analyzed by mass spectrometry.
The mass spectrometric analysis was performed using a nano-LC column coupled online to a QStarXL mass spectrometer (Applied Biosystems). Peptides were loaded onto a 75 cm × 10 cm, 3-mm fused silica C18 capillary column, and mobile phase elution was performed using buffer A (0.1% formic acid in 2% acetonitrile/98% Milli-Q water) and buffer B (0.1% formic acid in 98% acetonitrile/2% Milli-Q water). The peptides were eluted using a gradient from 2% buffer B to 100% buffer B over 90 min at a flow rate of 300 nl/min. The LC eluent was directed to an ESI source for Q-TOF-MS analysis. The mass spectrometer was set to perform information-dependent acquisition (IDA) in the positive ion mode for a selected mass range of 300–2,000 m/z. Peptides with +2 to +4 charge states were selected for tandem mass spectrometry, and the time of summation of MS/MS events was set to 3 s. The two most abundantly charged peptides above a 10-count threshold were selected for MS/MS and were dynamically excluded for 60 s with a ±50-mmu mass tolerance.
The raw data were analyzed using Proteome Discoverer 1.4 (Thermo Fisher Scientific). The software was connected to a Mascot Search Engine server version 2.2.4 (Matrix Science, London, UK) and to a Sequest Search Engine version 28.0 (Thermo Fisher Scientific). The confidence value for each peptide was calculated based on the agreement between the experimental and theoretical fragmentation patterns. Each protein was assigned a confidence score (0% to 100%) based on the confidence scores of its constituent peptides based on unique spectral patterns. Proteins with confidence scores of greater than 90% and with at least 1 peptide of 95% identification confidence were used for further quality control and differential expression analyses. Each protein also received quantitative scores for each of the eight-iTRAQ tags to calculate the relative expression levels. In this experiment, the relative expression levels of proteins in different samples were calculated using a normal sample as the reference sample.
The theoretical pI values and molecular weights (MWs) of the identified proteins were obtained from the UniProt protein sequence database. Functional enrichment analysis was performed using Gene Ontology (GO) (http://www.geneontology.org/), and GO annotation was applied to describe the functions of the differentially expressed proteins, which were classified into three major categories: cellular component, molecular function, and biological process . Pathway analysis was performed by KEGG mapping. Both assays proved statistically significant with p-values of less than 0.01 and 0.05, respectively.
Proteins were extracted from cells in log-phase growth and were separated using SDS–PAGE. A western blot analysis was then performed according to standard procedures. Briefly, total proteins were resolved by 10% SDS-PAGE and then transferred to nitrocellulose membranes. After incubating with primary antibodies at 4°C overnight, the nitrocellulose membranes were then washed three times with Tris-buffered saline containing Tween-20 (TBST) and incubated with horseradish peroxidase-conjugated secondary antibodies (1:2,000; Santa Cruz, USA) for 2 h at room temperature. The membranes were then washed again in TBS-T and visualized using an Enhanced ChemiLuminescence Kit (ECL-Kit, Santa Cruz, USA). Anti-CTBP2 and anti-EIF4E primary antibodies used for western blotting (1:500 dilutions; Abcam, USA), and an anti-β-actin antibody (Beyotime, China) was used as an internal reference. The experiments were repeated three times.
GraphPad Prism and image J software were used for data analysis. The results are presented as the mean ± standard deviation. Student's t-test was performed to evaluate differences between the western blotting analysis results. P-values of less than 0.05 were considered statistically significant.
In the present study, the localization of GMBP1 in multi-drug resistant gastric cells SGC7901/ADR and SGC7901/VCR was demonstrated by immunofluorescence staining and flow cytometric assays. As shown in the immunofluorescence staining assay, in both cell lines, positive staining was mainly located in the cytoplasm and was observed as a green color in the FITC-GMBP1 group; in contrast, the PBS group exhibited no staining (Figure 1(A,B)). Similarly, flow cytometry analysis showed higher fluorescence intensity for FITC-GMBP1 bound to SGC7901/ADR and SGC7901/VCR cells compared to the negative control FITC-URP group (Figure 1(C)). These results demonstrate that GMBP1 and its receptor GRP78 were located in the cytoplasm of gastric cancer cells but not in the control group.
Subcellular localization of GMBP1 and its receptor GRP78 in SGC7901/ADR and SGC7901/VCR. (A-B): a, d, g, j: The cytoplasmic localization of internalized GRP78 (green). b, e, h, k: Nuclear staining with 4, 6-diamidino-2-phenylindole (DAPI; blue). c, f, i, l: Merged images showing the relationship between GRP78 and the nucleus. (C): The internalization of the GMBP1 peptide into SGC7901/ADR and SGC7901/VCR cells. FITC-GMBP1 bound to SGC7901/ADR and SGC7901/VCR cells exhibited higher fluorescence intensity than the negative control FITC-URP group.
To explore the role of GRP78 in the internalization of the GMBP1 peptide into multi-drug resistant gastric cells, the specific downregulator GRP78 siRNA (siGRP78) and control siRNA (siCtrl) were transfected into SGC7901/ADR and SGC7901/VCR cells. Western blot and RT-PCR analyses showed that the transfection of SGC7901/ADR and SGC7901/VCR cells with the specific GRP78 siRNA resulted in a marked inhibition of GRP78 protein expression and decreased mRNA levels compared to cells transfected with the control siRNA (p < 0.01) (Figure 2(A,B)). An immunofluorescence staining assay showed that the control group incubated with FITC-GMBP1 did exhibit green staining (Figure 2(C, D)); the same results (data not shown) were observed using the GRP78 inhibitor. These results suggest that GMBP1 was internalized into the multi-drug resistant gastric cells and that this internalization was receptor mediated.
Internalization of the GMBP1 peptide into SGC7901/ADR and SGC7901/VCR cells. (A): Relative expression of GRP78 in SGC7901/ADR cells and SGC7901/VCR cells transfected with control-siRNA or GRP78-siRNA, which were confirmed western blot analysis. The values represent the means from three separate experiments, and the error bars represent the SEM (*P < 0.01). (B): The relative mRNA level of GRP78 in SGC7901/ADR and SGC7901/VCR cells. (C, D): a, d, g, j: The cytoplasmic localization of FITC-GMBP1 (green). b, e, h, k: Nuclear staining with 4, 6-diamidino-2-phenylindole (DAPI; blue). c, f, i, l: Merged images showing the relationship between GMBP1 and the nucleus.
To further characterize the mechanism of GRP78-mediated GMBP1 internalization in multi-drug resistant gastric cells, a double immunofluorescence staining assay was used. The effects of GRP78-mediated GMBP1 internalization on the uptake of Alexa Fluor 594-transferrin by the multi-drug resistant gastric cells are shown in Figure 3. Cells were doubly labeled with FITC-GMBP1 (green) and Alexa Fluor 594-transferrin (red) under control conditions at 37°C for 30 min; both FITC-GMBP1 and Alexa Fluor 594-transferrin were internalized, and FITC-GMBP1 was observed on the cell surface and in the cytoplasm (Figure 3(a, i)), whereas transferrin was observed primarily in the cytoplasm (Figure 3(b, j)). The labeled proteins were found to colocalize in the cytoplasm and perinuclear regions of the cells (Figure 3(d, l)). Furthermore, when chlorpromazine (CPZ), an inhibitor of clathrin-dependent endocytosis [31,32], blocked transferrin uptake, the red fluorescence of Alexa Fluor 594-transferrin was barely detectable (Figure 3(f, n)), and the green fluorescence of FITC-GMBP1 was also greatly reduced (Figure 3(e, m)). These results showed that the GRP78-mediated internalization of GMBP1 occurred through a clathrin-independent, transferrin-related pathway.
The mechanism of GRP78-mediated GMBP1 internalization into SGC7901/ADR and SGC7901/VCR cells. (A, B): a, i: FITC-GMBP1 observed on the cell surface and in the cytoplasm. b, j: Alexa Fluor 594-transferrin observed primarily in the cytoplasm. e, m: Internalization of FITC-GMBP1 was strongly decreased after blocking the uptake of Alexa Fluor 594-transferrin. f, n: Chlorpromazine largely blocked the uptake of Alexa Fluor 594-transferrin. c, g, k, o: Nuclear staining with 4, 6-diamidino-2-phenylindole (DAPI; blue). d, h, l, p: Merged images showing the relationship between GMBP1 and transferrin.
Our goal was to identify differentially expressed proteins that are related to MDR in GC and subsequently, to validate a subset of these proteins. We used cells from the multidrug-resistant gastric cell lines SGC7901/ADR and SGC7901/VCR for this study, and a schematic flow of the iTRAQ method used is shown in Figure 4. To increase the coverage of protein identification and/or the confidence in the data generated, proteins from these cell lines were labeled with iTRAQ reagents (the 113 tag for cell line SGC7901/ADR and the 115 tag for GMBP1-treated SGC7901/ADR cells). Thus, the ratio of labels 115 and 113 would indicate the relative abundance of MDR-related proteins. Similarly, proteins from these cell lines were also labeled with iTRAQ reagents (the 118 tag for cell line SGC7901/VCR and the 119 tag for GMBP1-treated SGC7901/VCR cells). Again, the ratio of labels 119 and 118 would also indicate the relative abundance of MDR-related proteins. To examine the biological reproducibility of the results, duplicate protein samples were obtained from both control and GMBP1-treated groups in two independent experiments. The iTRAQ analysis was performed in double-duplex style.
The flow chat of iTRAQ method and representative MS/MS spectrum of target proteins. (A): A schematic flow of the iTRAQ method. (B): A representative MS/MS spectrum showing CTBP2 and EIF4E peptides.
All the unique proteins were identified (false discovery rate < 1%) in the two biological replicates, and linear regression analyses were performed to examine the biological reproducibility of the results. Although the relative quantification analysis conducted using Protein Pilot 3.0 software includes statistical analysis, most methods are prone to technical variation; therefore, we included an additional 1.5-fold change and a 0.8-fold change cutoff for all iTRAQ ratios to reduce false positives for the selection of differentially expressed proteins. In total, 143 proteins were differentially expressed in the GMBP1-treated SGC7901/ADR cells compared with the SGC7901/ADR cells: 95 proteins were upregulated and 48 were downregulated (Additional file 1). For the SGC7901/VCR cells, 217 proteins were expressed differently following GMBP1 treatment compared to the control cells: 129 were upregulated, and 88 were downregulated (Additional file 2). Protein properties, including pI, molecular weight (MW), and number of residues, were calculated using PEPSTATS in EMBOSS. The grand average hydropathy (GRAVY) values were calculated as the arithmetic mean of the sum of the hydropathic indices of each amino acid.
The functional classification of all 3,752 proteins that were identified in the GMBP1-treated SGC7901/ADR cells is presented in Figure 5A. Proteins were cataloged according to biological processes (BPs), molecular functions (MFs), and cellular components (CCs) according to the GO database. The proteins representing BPs included cellular nitrogen compound metabolic processes (16%), biosynthetic processes (15%), small molecule metabolic processes (12%), signal transduction (10%), transport (9%), response to stress (8%), cellular protein modification processes (8%), anatomical structure development (8%), nucleobase-containing compound catabolic processes (7%) and cell differentiation (7%). The MFs of the proteins were classified, and the largest groups were found to be involved in binding (77%), oxidoreductase activity (7%), ATPase activity (4%), enzyme regulator activity (4%), kinase activity (4%) and transmembrane transporter activity (4%). The proteins representing CCs were classified as cytoplasm (17%), nucleus (17%), protein complex (12%), co-organelle (10%), extracellular region (9%), cytosol (9%), intracellular (8%), mitochondrion (7%), plasma membrane (6%) and cytoskeleton (5%).
Classification of the identified proteins by GO database. (A): Classification of the proteins that were identified in GMBP1-treated SGC7901/ADR cells. Biological processes (BPs), cellular components (CCs), and molecular functions (MFs) of all identified proteins, as classified according to the GO database. (B): Classification of the proteins that were identified in GMBP1-treated SGC7901/VCR cells. Biological processes (BPs), cellular components (CCs), and molecular functions (MFs) of all identified proteins, as classified according to the GO database.
The functional classification of all 3,749 proteins identified in the GMBP1-treated SGC7901/VCR cells is presented in Figure 5B. Proteins were categorized as BPs, MFs, and CCs according to the GO database. BP proteins represented cellular nitrogen compound metabolic processes (17%), biosynthetic processes (16%), signal transduction (11%), cellular protein modification processes (9%), small molecule metabolic processes (9%), transport (8%), anatomical structure development (8%), response to stress (8%), cell differentiation (7%) and nucleobase-containing compound catabolic processes (7%). MF proteins were also classified, and the largest groups were found to be involved in binding (69%), cytoskeletal protein binding (7%), kinase activity (6%), enzyme regulator activity (6%), ATPase activity (4%), nucleic acid binding transcription factor activity (4%) and oxidoreductase activity (4%). Identified CC proteins were classified as belonging to the nucleus (19%), cytoplasm (17%), protein complex (13%), organelle (9%), intracellular (9%), extracellular region (8%), cytosol (8%), plasma membrane (6%), cytoskeleton (6%) and nucleoplasm (5%).
The differentially expressed proteins were further defined based on KEGG (http://www.genome.jp/kegg/). The proteins were mapped to KEGG pathways based on their KEGG gene ID. The proteins differentially expressed in GMBP1-treated SGC7901/ADR and SGC7901/VCR cells are involved in 38 KEGG pathways and 79 KEGG pathways, respectively (results not shown). All pathways were statistically significant and based on research. As shown in Figure 5C, we used hypergeometric distribution in the enrichment analysis to prioritize these pathways, and the top ten KEGG pathways were summarized for both cell lines. The results (Figure 6(A)) indicated ten significant (p < 0.05) pathways in the GMBP1-treated SGC7901/ADR cells, including pathways for HTLV-I infection, Fanconi anemia, Influenza A, tight junctions, proteoglycans in cancer, Notch signaling, Jak-STAT signaling, N-glycan biosynthesis, adherens junctions and Wnt signaling. Figure 6(B) shows the ten most significant pathways in the GMBP1-treated SGC7901/VCR cells, which included pathways for adrenergic signaling in cardiomyocytes, PI3K-Akt signaling, ubiquitin-mediated proteolysis, tight junctions, HTLV-I infection, AMPK signaling, oxytocin signaling, dopaminergic synapses, gastric acid secretion and glutathione metabolism. Representative pathways associated with gastric cancer were investigated, including the Notch, Wnt, p53, PI3K-Akt and calcium signaling pathways. Further research is required to verify the proposed link between these pathways and GC MDR.
Classification of the identified proteins by KEGG database. (A): The ten most significant KEGG pathways in GMBP1-treated SGC7901/ADR cells. (B): The ten most significant KEGG pathways in GMBP1-treated SGC7901/VCR cells.
Among the proteins that were differentially regulated in the GMBP1-treated SGC7901/ADR and SGC7901/VCR cells, those that were the most downregulated in the two cell lines, EIF4E and CTBP2, are involved in the PI3K/AKT and the Notch and Wnt signaling pathways. To validate the effects of GMBP1 on several of the identified targets, a western blotting assay was performed. As shown in Figure 7, the expression levels of EIF4E and CTBP2 proteins were clearly downregulated (p < 0.01). This trend is similar to that observed for protein expression according to the iTRAQ analysis.
A representative western blot analysis of CTBP2 and EIF4E expression in the four cell lines comparing SGC7901/ADR and SGC7901/VCR cells and GMBP1-treated SGC7901/ADR and SGC7901/VCR cells. The values represent the means from three separate experiments, and the error bars represent the SEM (*P < 0.01).
Resistance to chemotherapy is a recurring issue for all cancer types, and the development of MDR is a major obstacle to the effective treatment of gastric cancer . However, the mechanism of MDR remains obscure. To study MDR in gastric cancer, we used as cellular models two drug-resistant cell lines, SGC7901/VCR and SGC7901/ADR, which were derived from the human gastric cancer cell line SGC7901 by stepwise selection in vitro using adriamycin and vincristine, respectively. These cell lines have been widely used as in vitro models for the study of MDR in gastric cancer [34-37]. Small molecules and short peptides have been considered for use in novel research on MDR because they exhibit many advantages, including rapid blood clearance, high tissue penetration and diffusion, non-immunogenicity and a high affinity for target tumor cells [9-11]. For example, in a previous study involving many peptides, our research team identified two peptides that bind specifically to GC vascular endothelial cells: GEBP11 and GX1. GX1 was also found to inhibit tumor growth. Using a phage display approach, we investigated the GMBP1 peptide, which specifically binds to the surface of gastric cancer MDR cells and exhibits the potential to be internalized into these cells and reverse the gastric MDR phenotype. Subsequently, GRP78 was identified as a receptor for this peptide . The success of our previous work and the known advantages of these short peptides encouraged us to study the effects of GMBP1 on GC MDR.
To further investigate the targeted binding sites and the subcellular localization of the GMBP1 and GRP78 peptides, we investigated the underlying internalization mechanism of GMBP1 using immunofluorescence staining combined with FACS. The results indicated the localization of GMBP1 and its receptor GRP78 in the cytoplasm of gastric cancer cells. In addition, we found that the internalization of GMBP1 into multidrug-resistant gastric cells was mediated by its receptor, GRP78. A double immunofluorescence staining assay demonstrated that the uptake of GMBP1, which was mediated by GRP78, occurred through a clathrin-independent transferrin-related pathway.
MDR is a multifactorial and multistep process, and a variety of biological factors are involved in GC MDR. Therefore, a global view of the interconnectivity of signaling proteins and their actions is critically important for the successful reversal of GC MDR. To date, proteomics analyses have proved to be powerful tools for identifying biological markers and for estimating biological networks . Proteomic methods have also been used to study the mechanisms of GC MDR. For example, Hu et al. revealed that MVP, one of the differentially expressed proteins found in our study, was highly expressed in SGC7901/VCR, and MDR was verified using iTRAQ-based proteomics . In the present study, the iTRAQ-based method was used to analyze the molecular mechanisms occurring in GMBP1-treated multidrug-resistant gastric cells SGC7901/ADR and SGC7901/VCR. To validate the reliability of this technology, the iTRAQ results were corroborated by conducting a literature review (in PubMed) and by western blot analysis. We determined that 83.6% of the affected proteins are also associated with other cancers, indicating that our data are consistent with those of other researchers. The differentially expressed proteins that were identified exhibited by a wide range of molecular weight (MW), pI, and GRAVY values. Moreover, bioinformatics analysis revealed that these proteins are involved in many BPs in GMBP1-treated SGC7901/ADR and SGC7901/VCR cells, including cellular nitrogen compound metabolic processes, biosynthetic processes, small molecule metabolic processes, signal transduction, transport, response to stress, cellular protein modification processes, anatomical structure development, nucleobase-containing compound catabolic processes and cell differentiation. In GMBP1-treated SGC7901/ADR cells, these proteins were found to be involved in 38 KEGG pathways that are connected with each other to form a network. Furthermore, the proteins identified in GMBP1-treated SGC7901/VCR cells are involved in 79 KEGG pathways. These findings illustrate that multiple mechanisms can cause drug resistance in gastric cancer cells and that these mechanisms might partially contribute to chemotherapeutic resistance during gastric cancer treatment.
Deregulation of the PI3K/AKT pathway plays a crucial role in the regulation of multiple cellular functions, including cell growth, proliferation, metabolism, and angiogenesis. Notably, numerous reports have implicated the PI3K-Akt signaling pathway in gastric cancers . Among the differentially expressed proteins identified, the level of eukaryotic translation initiation factor 4E (EIF4E) was markedly downregulated in both GMBP1-treated cell lines, which was confirmed by western blot analysis. As a member of the PI3K/AKT pathway, EIF4E has been identified as an oncogene that plays a role in many malignant diseases, including GC. Silencing of EIF4E was found to slow proliferation and arrest the cell cycle in G0/G1 phase in larynx, stomach, and breast cancer cells [39-41]. These findings indicate that EIF4E expression might represent a molecular target for cancer therapy and lead to the assumption of a possible role for EIF4E in MDR. Combining our present and previous work, we hypothesis that the GMBP1 peptide modulates gastric cancer MDR by targeting GRP78 and that the expression of GRP78 modulates the expression of EIF4E and MDR1 through the PI3K/AKT pathway (as shown in Figure 8). Our study also showed that PPP2R2A, PRKAA1, and PCK2 were overexpressed in GMBP1-treated SGC7901/VCR cells. These proteins are also members of the PI3K/AKT pathway, and their function in GC MDR merits further investigation.
Schematic drawing of the mechanism of the GMBP1 peptide in modulating gastric cancer MDR by targeting GRP78.
The Notch signaling pathway and the Wnt signaling pathway are increasingly recognized as critical for the regulation of drug resistance. C-terminal binding proteins (CTBPs) are transcriptional corepressors that mediate the Notch and Wnt pathways, among others. Of the proteins identified using iTRAQ, C-terminal binding protein 2 (CTBP2) was downregulated in both GMBP1-treated cell lines; furthermore, the protein level of CTBP2 was lower in the GMBP1-treated SGC7901/ADR and SGC7901/VCR cells than in the control cells. CTBPs interact with many DNA-binding transcription factors, including mediators of Wnt, BMP, and Notch signaling [42,43], GATA factors , and regulators of several key processes, including myogenesis , vascularization , apoptosis, and cell adhesion . Paliwal et al. identified the CTBP2 transcription regulator as an ARF-binding protein and observed that the targeting of CTBP by ARF results in p53-independent apoptosis . Furthermore, Paliwal et al. showed that CTBP might promote tumor proliferation . Recent studies have shown that MDR phenotype acquisition is often associated with increased tumor invasion and metastasis . MDR not only prohibits effective chemotherapy but also exacerbates the metastatic symptoms of cancer patients; CTBP proteins, which had not previously been associated with MDR, are now shown to play a role in the development of MDR.
Transcription factors and proteins related to signal transduction were found to be differentially expressed between GMBP1-treated and untreated multidrug-resistant gastric cells but have not been associated with MDR to date. The correlation between these proteins and MDR in gastric cancer cells will be the subject of future study.
In conclusion, GMBP1 exhibited significant potential to reverse GC MDR. Our results showed that iTRAQ is a powerful technique for performing quantitative proteome analysis in relation to drug resistance, and a large number of differentially expressed proteins were identified in this study. Our results further confirmed that the GMBP1-GRP78 component plays an important role in the drug-resistant phenotype of gastric cancer cells. GMBP1 may therefore represent a novel MDR reversal agent for the management of GC. Other mechanisms that act against GC MDR in the GMBP1-GRP78 module should be investigated further. The data obtained will prove useful for the study of the mechanism of MDR in human GC and provide new clues for investigating MDR in other tumors.
Xiaojuan Wang and Yani Li contributed equally to this work.
This work was supported by the National Natural Science Foundation of China (Nos. 81472778,81272516, 81090273, 81090270, 30900674, 30973428) and the Clinical New Techniques Project of Xijing Hospital (No. XJGX13LZ02).
Additional file 1: Table S1. iTRAQ analysis of proteins that were differentially expressed between GMBP1-treated SGC7901/ADR (iTRAQ 115) and SGC7901/ADR (iTRAQ113) cells.
Additional file 2: Table S2. iTRAQ analysis of proteins that were differentially expressed between GMBP1-treated SGC7901/VCR (iTRAQ 119) and SGC7901/VCR (iTRAQ118) cells.
XW, SL, BW and JD designed the study and experimental protocols. SH and YZ directed the proteomic research. XW and YL wrote the first draft of the manuscript together. GX and ML participated in FACS staining and analysis. XW, YN and LX completed the protein identification and bioinformatics analysis. XW participated in protein identification. LL and YL finished the WB experiments. All authors have read and approved the final manuscript.


A new, twice-yearly symposium series brings noted scholars in various stages of their careers together with faculty, graduate students, and undergraduates in the Department of History.
The Mary Clare Reynolds Joyce Symposium series is funded through the Mary Clare Reynolds Joyce Endowment for Excellence in the Department of History, a gift from Robert (ND '53) and Sheila Berner, made in memory of their niece. The series will engage a broad spectrum of participants and will be designed to maximize the exchange of ideas between the speakers, faculty, and students.
The initial symposium, held in September, honored Thomas A. Brady, Jr., Peder Sather Professor Emeritus and Professor of the Graduate School at University of California, Berkeley. Brady, a 1959 Notre Dame graduate, is a leading scholar on the German Reformation. His career-summarizing book, German Histories in the Age of Reformations, 1400-1650, was published last year by Cambridge University Press.
The department also invited H.C. Erik Midelfort, Charles Julian Bishko Professor Emeritus at the University of Virginia; Sigrun Haude, associate professor at the University of Cincinnati; and John Frymire, associate professor at the University Missouri at Columbia, to reflect on Brady's work and dialogue with him.
The four Reformation scholars spent two days on campus, interacting with faculty, meeting individually with graduate students, and visiting classrooms.
Daniel Graff, who teaches the one-credit Exploring History Beyond the Classroom course for history majors, says undergraduates also benefit from the symposium series.
The plan is to arrange a set of similar interactions around each of the events in the series. "We will move it around the various areas of the department," Noble says, adding that Americanists are organizing the symposium for the spring 2011 semester.
The events are open to graduate and undergraduate students, faculty and the community.


text:
Rebecca McClure
Originally from Alberta, Rebecca McClure began her singing career at a very young age, crooning cowboy songs while accompanied by her dad on guitar and harmonica. The youngest of five children, she frequently performed in religious venues with her parents and sisters, learning how to harmonize by ear by age 7.
At one point in her mid-teens, she traded off piano lessons for voice lessons and fell in love with classical repertoire and solo singing.
In university, Rebecca continued with piano, even though her academic studies were primarily focussed on sciences and humanities, and she taught piano as she worked through the advanced RCM material.
She moved to the West Coast in the mid-90's to attend graduate school at UBC. Since then, through private voice lessons and participation in different choral opportunities, Rebecca has managed to keep music a big part of her life while juggling family, career, and other artistic pursuits.
Currently studying voice with Ingrid Attrot at the VCM and also singing with CapriCCio Vocal Ensemble, Rebecca is thrilled to add this exciting Winterreise Project to her roster of musical activities.
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Revolution Workbook with lesson summary, scriptures, questions and charts to help you apply the video lessons available through WeighDown All Access! Select Your format - PDF Download for your computer/mobile/tablet devices or have a softback copy shipped to you.
from Colorado Over the top helpful!!
This workbook that accompanies the Revolution videos and audios is a fantastic resource!! It makes me really think about my heart and helps me tell myself the truth so I can break free from being overweight!


In this video we're making a basic pepperoni pizza - I am hoping to add a follow-up video focusing on a few different types of toppings, but for this tutorial we're staying simple and classic.
I'll be showing you the materials you'll be needing; exact shades of paint, pastel and colors of clay used. I will not be showing the exact amounts of each clay color as I always eyeball it when mixing.


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What we do — build 360-degree communities in specialty therapeutic areas.
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Annodyne Health's board of advisors helps us craft strategies and pressure test our ideas for the real world. And we have a successful history building proprietary performance measurement platforms, custom mobile apps and interactive visual aids. When combined, we deliver proven health care marketing solutions powered by leading-edge technology solutions.
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text:
Q: Headset button double click in android I used this code for detecting single and double click for headset button in my broadcast receiver:
int d = 0;
@Override
public void onReceive(Context context, Intent intent) {
    String intentAction = intent.getAction();
    if (!Intent.ACTION_MEDIA_BUTTON.equals(intentAction)) {
        return;
    }
    KeyEvent event = (KeyEvent) intent
            .getParcelableExtra(Intent.EXTRA_KEY_EVENT);
    if (event == null) {
        return;
    }
    int action = event.getAction();
    switch (event.getKeyCode()) {
        case KeyEvent.KEYCODE_HEADSETHOOK:
            if (action == KeyEvent.ACTION_DOWN) {
                d++;
                Handler handler = new Handler();
                Runnable r = new Runnable() {
                    @Override
                    public void run() {
                        Toast.makeText(context, "single click!", Toast.LENGTH_SHORT).show();
                        d = 0;
                    }
                };
                if (d == 1) {
                    handler.postDelayed(r, 500);
                } else if (d == 2) {
                    d = 0;
                    Toast.makeText(context, "double click!", Toast.LENGTH_SHORT).show();
                }
            }break;
    }
    abortBroadcast();

}

but it just detects single click. Two single click instead of double click. Where's the problem?

A: The correct solution:
static int d = 0;
@Override
public void onReceive(Context context, Intent intent) {
    String intentAction = intent.getAction();
    if (!Intent.ACTION_MEDIA_BUTTON.equals(intentAction)) {
    return;
    }
    KeyEvent event = (KeyEvent) intent
        .getParcelableExtra(Intent.EXTRA_KEY_EVENT);
    if (event == null) {
    return;
    }
    int action = event.getAction();
    switch (event.getKeyCode()) {
    case KeyEvent.KEYCODE_HEADSETHOOK:
        if (action == KeyEvent.ACTION_DOWN) {
            d++;
            Handler handler = new Handler();
            Runnable r = new Runnable() {

            @Override
            public void run() {
                    // single click *******************************
                    if (d == 1) {
                                Toast.makeText(context, "single click!", Toast.LENGTH_SHORT).show();
                    }
                    // double click *********************************
                    if (d == 2) {
                        Toast.makeText(context, "Double click!!", Toast.LENGTH_SHORT).show();
                    }
                    d = 0;
            }
            };
            if (d == 1) {
                    handler.postDelayed(r, 500);
            }
        }break;
   }
   abortBroadcast();
}

redpajama_set_name: RedPajamaStackExchange


text:
Not Being Heard
by Anne Kelly
(British Columbia, Canada)
I am a woman of 58 who grew up on northern Canadian military bases. I was a child of 8 when I came to Canada with my parents and two sisters. At the age of 11, I was molested by the DND (Department of National Defence) employed principal of the school where I attended for two years. This military compound had no media or television, and every house was furnished like the others.
My younger sister and I were abused my two other military men, which began when we arrived in Canada; I was 8 and Mary was 7. In the military, you often meet people from other bases. It was like a nightmare come true in the 50's and 60's to encounter such men as had abused us before. Although this was reported at the time in the 60's; it took me until the 90's to look at how this had affected my life: in a way that I will never recover.
In 1998, two men from the DND military police came to my home in Duncan, British Columbia. They interviewed me and they left. I never heard from them. Ten days later, I called their office in Edmonton. They informed me that the "statute of limitations" had run out in one case. In the 2nd case, I was asked whether or not "do you really want to charge a 92-year-old?" The 92-year-old was the principal of the Protestant DND School in Parent, Quebec. This man tormented me from the age of 11 - 13 everyday for two years. His wife, Helen, was my teacher. It is the responsibility of the DND to provide schools whereby the principal is not a child molester! In the 3rd case, I was told that the RCMP in (location not disclosed)...may be interested.
Today, when I ask for a copy of the review and the decision, I am told that the papers are in the vault and I will have to go through some procedure to look at them. How many others were molested by these three men?
Thank you for this forum which helps me to speak, at least and at last, about my experience as an immigrant child disguised by white skin and blue eyes. Thank you for this opportunity to be heard.
Email addresses, phone numbers, home addresses AND website/blog URLs in visitor comments are strictly prohibited.
Join in and write your own page! It's easy to do. How? Simply click here to return to Child Abuse Commentary.


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Guardian Sport Network
How Nice beat all the odds – and PSG – to challenge for the Ligue 1 title this season
Nice finished 33 points behind Paris Saint-Germain last season but, after a 3-1 win over the champions on Sunday, they are still challenging for the title
By Adam White and Eric Devin for Get French Football News
Adam White and Eric Devin
Mon 1 May 2017 06.00 EDT Last modified on Wed 21 Feb 2018 08.16 EST
Mario Balotelli scored his 14th goal of the season in Nice's 3-1 win over PSG. Photograph: Yann Coatsaliou/AFP/Getty Images
"It was a great match, one that completes our season. Once more, this isn't down to luck, we know this. We know our level and the progress we've made. It is greatly satisfying for the players and the fans." OGC Nice's president, Jean-Pierre Rivère was understandably proud of his team after a 3-1 home win over Paris Saint-Germain. Sublime goals from Mario Balotelli and Ricardo Pereira caught the eye and young goalkeeper Yoan Cardinale was superb, but Nice are focused on the bigger picture.
A combination of Monaco all but being assured of the Ligue 1 title and a near-complete meltdown by Paris Saint-Germain at the end of the match may overshadow the result, but make no mistake, Nice's victory over the defending champions should be read as a valedictory statement for the hosts and the progress they have made under Rivère. Nice are still all but out of the title race themselves and some will view their tumble down the table in 2017 as a weakening of their early resolve, but they are now unbeaten in 12 matches, something that would have seemed highly unlikely three months ago.
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Indeed, the last time the casual observer of French football heard from Nice was probably their 3-0 loss at Monaco in early February. Already out of Europe and both domestic cups at that point, the defeat completed a run of only one win in five in the league that took them from first to third. What had looked a charming story at the beginning of the season now looked to be slipping away, as the team struggled with a lack of depth against their well-heeled rivals. Most observers thought Nice had more to worry about from Lyon, in fourth, who had a game in hand and had just signed Memphis Depay.
After that defeat to Monaco, Nice lifted themselves off the canvas with an impressive 1-0 win over Saint-Étienne. There has been the odd draw in this unbeaten stretch, including away to Toulouse last Sunday, but the team's response in the face of adversity has been hugely impressive. They have endured season-ending injuries to midfield tyro Wylan Cyprien and then-leading scorer Alassane Pléa, as well as lengthy fitness problems concerning captain Paul Baysse, Ricardo Pereira, Valentin Eysseric, Younès Belhanda and Mario Balotelli. Add in Jean-Michaël Seri's stint at the Africa Cup of Nations and young centre-back Malang Sarr's dramatic loss of form, and Nice's form seems a minor miracle.
Much of the credit has to go to Lucien Favre, who has used an ingenious variety of tactics to manage these absences. The team started the season playing a 4-3-3 before moving to three at the back. More recently, in the absence of Cyprien, they have moved to a 4-2-3-1. Pereira, brought in as a right-back, has been used as an orthodox winger, which has allowed Seri to push further forwards. A defensive midfielder upon his arrival, the Ivorian has been a revelation this season in a more attacking role, with 10 assists and six goals.
Lucien Favre, who replaced Claude Puel as Nice manager last summer, barks instructions at his players against Paris Saint-Germain. Photograph: Sebastien Nogier/EPA
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Favre has also given expanded roles to a few players who were something of an afterthought in the season's early going. Along with Dante and Belhanda, Mario Balotelli was the big-name arrival in the summer. There was also some intrigue over the promising Cyprien and Arnaud Lusamba, but Arnaud Souquet was something of an afterthought. A former youth international for France, he was a product of Lille's academy who just two years ago found himself playing for amateur side Poiré-sur-Vie. Success there saw him earn a move to Dijon, then playing in Ligue 2, and he was a key part of that team's promotion last season.
Still, though, he was hardly expected to play more than a handful of matches this season, as Pereira was unquestionably first choice. When Pereira was forced to miss more than a month with a knee injury in January, Souquet, who had only played a handful of matches, stepped up. He has played every minute in Ligue 1 since being deployed as a makeshift centre-back for the team's trip to the Parc des Princes. Now played as a right-back, he is an impressive attacking threat, having contributed five assists this season.
Another former Ligue 2 player, Maxime Le Marchand has also been important for Nice since signing from Le Havre in 2015. Like Souquet, he seemed destined for bigger things at a young age, having won the Coupe Gambardella, France's youth cup competition, in a Rennes team that included Yacine Brahimi and Yann M'Vila. However, after failing to break into the first team, he was forced to move down a level. Initially a left-back, Le Marchand impressed at centre-back this campaign before suffering a recent hamstring injury that has ended his season, becoming an invaluable deputy for Baysse during the captain's recent spell on the sidelines.
Eysseric also falls into this category, contributing six assists and three goals in 2017 after starting just eight times in the first half of the season, but the pattern that should be apparent now is one of an exceptional recruiting apparatus. Admittedly, Chinese backing was key in Nice being able to pay the wages of Balotelli, Dante and Belhanda, but the real secret to the team's success lies with their scouting department. Nice did splash €5m on Cyprien this summer (a figure that seems a pittance after his season), but the combined cost of Seri, Le Marchand, Pléa, Souquet, Baysse and Dalbert was less than €6m.
One could argue that even those paltry fees are beyond the means of most of Ligue 1 clubs, but Nice have also sold important players in the recent past, bringing in handsome returns for Jordan Amavi and Nampalys Mendy. In this way, Nice, through their scouting department, headed by Serge Recordier, have not only seen success but built with an eye towards sustainability. Since Rivère joined in 2011, the club have placed their focus on the academy, building a new training centre and allowing themselves to sustain success even in the face of significant departures.
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As Cardinale told RTL recently: "Last summer, there was a lot of uncertainty in the dressing room after the departure of our two top scorers, [Hatem Ben Arfa and Valère Germain] our captain [Nampalys Mendy] and the coach. In the end, though, everything worked out great. We've accomplished something historic and we're not setting limits."
Yesterday's win is a bold affirmation of the club's over-arching strategy. As Nice prepare for what looks set to be a place in the Champions League play-off rounds, there will be tough decisions to make on Balotelli (out of contract), Belhanda (an option to buy is reported to be in the region of €9m) and Pereira, whose loan is set to expire. There are also questions surrounding the ability of Cyprien and Pléa to recover from serious injuries, but the more one considers Nice's situation, though, the more likely that the combination of Favre, Fournier and Recordier looks set to keep success within reach.
Ligue 1 talking points
• The consistent stream of new thrilling young players is perhaps the most exciting aspect of French football. Kylian Mbappé's compelling development over the last six months has been astonishing, but the form of one player who never truly lived up to his hype at a similar age has been just as brilliant as the teenage Monaco forward's displays this campaign. Florian Thauvin's swaggering hat-trick in Marseille's 5-1 demolition of relegation threatened Caen was perhaps the high-point of a stunning season. Despite Thauvin's arrogant, lazy reputation in England, his intensity, attitude and work-rate have been exemplary for Rudi Garcia's side throughout the campaign. So much so that he won a call up from Didier Deschamps in March and beat Mbappé to the Ligue 1 Player of the Month award. The 24-year-old admitted recently that he had questioned himself after his trying spell with Newcastle, but his confidence has returned with him to France to stirring effect. 15 goals scored places him 5th in Ligue 1's scoring charts and with coupled with his seven assists, Thauvin's importance to Marseille is clear, regularly outshining Dimitri Payet since his return from West Ham. But crucially for Thauvin it is his renewed faith in his undoubted ability that shines through in each of his performances. The nonchalance and effortless grace with which he took all three of his superb strikes at Sunday lunchtime and his unerring pace and intensity whenever he was in possession will have Garcia delighted that Marseille were forced into activating the player's €11m purchase option owing to a "games played" clause in the loan agreement with Newcastle.
• When Monaco centre-back Jemerson scuffed a clearance into the path of Toulouse's Ola Toivonen early in the second half at the Stade Louis II on Saturday afternoon, allowing the Swede to slot home for 1-0, the title race seemed to be shifting in PSG's direction. The first half was, by Monaco's standards, stilted and blunt and, despite Jardim's decision to sacrifice the Coupe de France (resting his entire team for the mid-week semi-final at PSG, which a B team string lost 5-0), Monaco looked tired. "A response of Champions" may be clichéd description of their comeback, but it began to feel like Monaco are now close to unstoppable on the domestic front when Kamil Glik slammed home a header to equalise seconds later. The effervescent Kylian Mbappé arrowed home to take the lead 15 minutes after, 22 goals in 20 starts for the teenager as his unbelievable form continues, while Thomas Lemar made sure Monaco managed their now customary three goals per Ligue 1 match and stayed three points clear of PSG. But perhaps it was in the 5-0 thumping Monaco received from PSG where Jardim made his most astute move of the week. Monaco have looked a little weary at time in recent months. Their comparatively small squad, busy schedule and all action style has begun to catch up with them. In contrast, since their humiliation in Barcelona, PSG have returned to their swaggering best in Ligue 1 and dispensed with an admittedly Fabinho-less Monaco in the League Cup final, 4-1. As a result, even if Jardim picked his strongest XI for the Coupe de France semi-final in the week, PSG would have been favourites at the Parc des Princes, where they are very strong. By sacrificing the Coupe de France, Jardim gave his first team a much needed rest and simultaneously covertly sidestepped a potentially confidence, momentum and energy draining defeat to their rivals; perhaps a key psychological move that could aid in what now looks like a title win for Les Monegasques.
Kylian Mbappé celebrates with Monaco team-mate Thomas Lemar against Toulouse. Photograph: Valery Hache/AFP/Getty Images
• End of season awards and shortlists are by definition a subjective business but as the French professional footballers body, the UNFP, released their various lists for their accolades this week, questions were raised. The UNFP have long be criticised for supposed big club bias, but the number of PSG nominations this year was surprising even by their standards. The perennial champions were represented on all four four-man shortlists, a more than curious situation after their comparatively poor season and fluctuating form. Edinson Cavani, as the league's top scorer, justifiably joins Bernando Silva and Alexandre Lacazette in contention for Player of the Year. However, the absence of Monaco's sublime midfield general Fabinho in favour of Marco Verratti is verging on ridiculous. Fabinho is Monaco's most important player and the difference in performances with him and without him both domestically and in Europe is stark. His power, drive and pace help him and partner Tiemoué Bakayoko to dominate midfield battles despite often being outnumbered while his range of passing, vision and creativity drive the side forward. Without him they would not be top of Ligue 1 and his omission is a baffling oversight. Verratti has been very good since Christmas and when on form is genuinely one of the best players on the planet, but when put next to Fabinho this season, there is only one winner. Jardim and Nice's Lucien Favre are obvious inclusions on the managerial list while Jocelyn Gourvannec's deft nurturing of a young Bordeaux side into the top 5 also earns him a deserved place alongside them.
Yet Unai Emery's appearance on the shortlist verged on the incomprehensible before his side's ill-disciplined, chaotic loss in Nice on Sunday evening, and now looks laughable. This season has been one of regression for PSG. Yes, they could still retain their treble and yes, this is a domestic award, so their humiliation in Barcelona theoretically should not affect the decision making. However, PSG for long spells have been brittle, lethargic and complacent and although their best form has seen them play at their swaggering best this has largely come at home against much weaker sides, the chasm in available resources and personnel to rival clubs and the 31-point margin of victory last season under Blanc are huge caveats any of the league's now second best side's return to form. More to the point, Emery has had very little discernible influence on displays, doing little more than making Laurent Blanc look good. This is not to eviscerate Emery, Monaco in their current form is something precious coaches did not have to deal with (although Lyon's title push in 2014/15 is not too dissimilar) and his CV is still a superb one, winning three consecutive Europa League title's with Sevilla but his credentials for being on this 2016/17 Ligue 1 shortlist are thin at best. Nantes' Sergio Conceição, Toulouse's Pascal Dupraz and even Lorient's Bernard Casoni all deserve far greater praise for the evolution of their teams this season, all unable to bring the likes of Lucas Moura, Blaise Matuidi and Javier Pastore off of their benches.
Ligue 1 results
Angers 1-2 Lyon, Monaco 3-1 Toulouse, Bastia 1-0 Rennes, Guingamp 0-2 St Étienne, Metz 2-1 Nancy, Montpellier 0-3 Lille, Nantes 1-0 Lorient, Caen 1-5 Marseille, Dijon 0-0 Bordeaux, Nice 3-1 Paris Saint-Germain
• This is an article from Get French Football News
• Follow Eric Devin, Adam White and Get French Football News on Twitter


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Review: Devil's Food Cake at Brighton Fringe (online)
Published on June 7, 2021 by Liz
Written, rehearsed and filmed in lockdown, Marcia Kelson's Devil's Food Cake sensitively shines a light on the impact of an eating disorder on one ordinary family. Seventeen-year-old Sophie (Eden Vansittart) is desperate to lose weight, even though to her mum Jenny (Lesley Ann Jones), dad Frank (David Jones) and sister Katie (Eliza Jones), she's looking more dangerously thin every day. With the support of therapist Jasmine (Caroline Salter), each member of the family adopts a different strategy to try and convince her to eat – but with Sophie well and truly under the spell of the persuasive Ana (Catherine Allison), are they too late to pull her back from the edge?
The play throws us straight into the heart of the story, with Sophie already fully committed to her extreme dieting regime, and her family very much aware that something's not right. The focus is never really on how they got to this point, but rather on where they go from here, and Kelson paints a moving picture of a loving family unit at risk of being pulled apart by a dangerous force they can't see and don't know how to defeat. Zoom productions come with some inevitable limitations, but in this case the format is used well, placing Sophie alone on one screen, while her mum, dad and sister sit together around the kitchen table on another, highlighting her isolation and the growing distance between them.
It's unclear at what stage in the timeline of events the family reached out to a therapist, but the presence of Jasmine allows us to gain an insight into the psychology of eating disorders, not only for those directly affected, but also for their loved ones. In fact it often feels Jasmine is there more for the audience's benefit than for the family's; she spends much of her time directly addressing the camera, and her tenuous animal analogies, while helpful to explain the theory to an impartial observer, repeatedly fall flat with her clients. It's only when she begins to engage on a more personal level that the family start to make some progress.
This isn't the only point where the play purposefully sets out to educate. While the part social media plays in promoting unhealthy body image may be obvious to most, there's also an interesting scene in which Katie explains to her dad the less obvious role of cookies (the computer kind), and the danger that in seeking help online, those affected by eating disorders may find themselves drawn into "support" groups of the wrong kind. This conversation feels a bit deliberate and does little to develop the story, but it does add value to the play in demonstrating how modern technology can simultaneously both help and harm in ways we might not have thought about.
Eden Vansittart gives a compelling performance as Sophie, a teenager caught between her affection for her family and her desperate fear of not living up to the ideal standards Ana demands from her daily. Ultimately this is her journey, and despite everyone's efforts to help, she alone can decide which path she wants to follow. But that's not as straightforward as it sounds, and Vansittart captures well the turmoil her character is experiencing, especially in a powerful final scene that takes the audience on an unexpected rollercoaster of emotion.
For those already familiar with the impact of eating disorders, Devil's Food Cake may hit a bit close to home – it comes with a clear trigger warning for good reason. But for those of us who know less, but want to understand more, it's a powerful and poignant portrait of the untold damage such conditions can cause to both health and relationships, and a fascinating (and depressing) insight into how the modern world we live in, even with all its medical and technological advances, may still only be making things worse.
Devil's Food Cake is available to watch online as part of Brighton Fringe until 27th June.
Previous Review: A Russian Doll at the Barn Theatre
Next Review: Queen Mab at the Actors' Church


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Green Gal: Environmentally Conscious Lifestyle
Allergy season is kicking in, and it's kicking me hard
By green_gal on March 12, 2008 at 9:05 AM
Spring is a wonderful time of year: the sun shines brightly, the birds chirp merrily, and I become permanently crippled by allergies until fall.
My allergies actually aren't as bad as they were when I was younger. When I lived in northern Indiana for a brief few years – let's not talk about it too much – I went to a school called Prairie Vista Elementary. The school had a prairie-type setting surrounding it, and we had something called Prairie Day in the spring. This involved setting us kids loose in the tall grasses to play prairie games. For me, it was nothing short of first degree murder by the school administration. Thanks, guys!
One of my good friends growing up had two cats. I recall some of our playdates ending in emergency evacuations from her house. As a result, I still have a strong, strong dislike of cats. I find them to be shady and calculating, in addition to being irritating to my sinuses. Hmm, there may be some underlying issues there…
Anyway – the cat thing is now manageable. The family I babysit for has two cats, but I pop a Claritin beforehand and am generally ok. There's no prairie around here, but when the cherry trees bloom in the quad – forget about it. Some trees are beginning to flower on my street right now, and I think they may be the cause of the recent onslaught of sneezing fits, nose-blowing, and general misery.
Here's the thing about managing my allergies: it sucks. Claritin is hit-and-miss, and it's expensive. Let's not even talk about Benadryl – it doesn't stand a chance. My nose is now red and raw from blowing it so much, and I hate having to breathe through my mouth when I fall asleep because my nose is so stuffed up.
So I really want to try this thing called a neti pot.
I first saw it on Oprah a while back on one of those "Ask Dr. Oz" episodes. It can also be described as a "nose bidet" – it looks like a little genie's lamp, and you fill it with warm, salty water. I kid you not, here's what you do: pour the water in one nostril and tilt your head so it comes out the other. It cleans out your sinuses!! Click here to see an audience member trying it.
Amy, an audience member who suffers from sinusitis, demonstrates the proper way to use a neti pot. "I thought it would feel like drowning, but it feels good," she says.
It may look strange, but Dr. Oz says this treatment may be more effective than medication. "The [ear, nose and throat] doctors who are specialists in this area will often say this is a better treatment than a lot of the other drugs that we try to offer folks, because it mechanically cleans out the problem," he says.
Oprah later followed up with the audience member and a Harpo staffer to see how the neti pot worked in the long run:
Since then, Amy says she and her husband have used the neti pot every day! "It is fantastic. I have not had one sinus headache. I haven't spent any money on any cold products," she says.
Rachel, a member of the Harpo staff, benefited from the neti pot, too! "It wouldn't be an exaggeration to say that it's been life-changing for me," she says. Rachel is a lifelong allergy sufferer, but since she began using the neti pot, her symptoms have eased up. "I can breathe at night. I'm not blowing my nose, you know, 30 times a day, which is my normal state of being for the last couple years," she says. "So thank you, Dr. Oz, because it was really a huge change for me and I'm very happy."
Honestly, if I saw this neti pot thing on an infomercial, I wouldn't believe it. But this is Oprah we're talking about. I need one!!
My mom has one from Whole Foods that she hasn't used yet, and I was going to try it when I was back home the other night. Sadly, I forgot to do it before I left.
I'm now on a neti pot mission – Oprah's Web site says they're available at health food stores and most drug stores. My dream is to acquire the neti pot, relieve my wretched allergies, and not have to buy expen$ive allergy medicine anymore. Remember when I said that I use way too much facial tissue? This is why! I want to stop that, too!
Has anyone ever used a neti pot? I'm interested in hearing about it!
Devon Mills


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EL SALVADOR PERSONALITIES
MI SANGRE
Home ᐳ El Salvador ᐳ Morazán, El Salvador
Morazán, El Salvador
GEOGRAPHIC PROFILES / 22 MAR 2021 BY INSIGHT CRIME EN
There is a minor presence of transnational organized crime in Morazán. Modest quantities of livestock and foodstuffs are smuggled over unmonitored roads along the department's border with Honduras.
There is a small illicit market for drugs that appears to be controlled by the Mara Salvatrucha (MS13) street gang.
MS13: The MS13's presence has been noted in many of Morazán's municipalities, including departmental capital San Francisco Gotera. Like elsewhere in El Salvador, the gang's members have access to light firearms such as handguns.
Criminal Economies
Arms Trafficking: There does not appear to be a significant black market for illicit firearms in Morazán. However, the thriving arms trafficking economies in San Miguel and La Unión may spill over into the department. The MS13 is an active buyer of illegal firearms, but the gang has only a minor presence in the department and does not appear to be involved in major turf wars with other criminal groups.
Cocaine: There is no evidence of a significant cocaine economy in Morazán, though there are minor seizures of cocaine destined for local consumption. The MS13 appears to be involved in small-scale cocaine dealing.
Cannabis: There is no reported production of marijuana in Morazán. There have been some small cannabis seizures, suggesting there is a modest local consumption market that may involve the MS13.
Human Trafficking: Human trafficking appears to be a small criminal economy in Morazán. There are some reports of children being subjected to forced labor and sexual exploitation; the latter may occur in brothels, which double up as retail spots for drugs.
Human Smuggling: We estimate that just over 1,000 Salvadoran migrants were returned to Morazán from the US and Mexico in 2019. Given the price of hiring a smuggler in the area (roughly $10,000), this appears to be a lucrative criminal economy, reaching millions of dollars. In addition to smugglers, individuals known as "comisionistas" (comission agents) purportedly operate in the department, seeking out new clients for a fee of a few hundred dollars.
Extortion: Morazán has relatively low extortion rates compared to other parts of El Salvador. The use of security cameras in departmental capital, San Francisco Gotera, appears to have helped with policing extortion rackets.
Contraband: There is some evidence of contraband smuggling through Morazán, usually involving individual, small-time traders who transport livestock and foodstuffs between El Salvador and Honduras.
Sources: This profile is based on a field investigation in Morazán and research in San Salvador, where InSight Crime interviewed high-ranking municipal officials in San Francisco Gotera, in addition to current and former national police officials. InSight Crime also drew from information provided by the Government of El Salvador, the Economic Ministry's General Directory of Statistics and Censuses (Dirección General de Estadística y Censos), police extortion data, the United Nations Office on Drugs and Crime (UNODC), El Salvador-based Diálogos think tank, and local press.
GEOGRAPHIC PROFILES
Santa Bárbara, Honduras
Santa Bárbara sees its fair share of narcotics seizures and has played host to major drug trafficking groups such as…
Chaco, Argentina
GEOGRAPHIC PROFILES / 15 MAR 2021
Chaco is a transit corridor for illicit products. Most drugs or contraband smuggled into Argentina in the Formosa province crosses Chaco…
Ñeembucú, Paraguay
Despite boasting one of Paraguay's lowest murder rates, Ñeembucú is an important transit point for marijuana heading to Argentina.