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capricornus-rex · 5 years ago

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Playing Pretend (4)

Requested by: @calkesttiss | Prompt:

Hi! I just watched isi & ossi (rich girl and poor boxer boy AH) on netflix and now i cant stop thinking about cal and fake dating. Do with that what you will 😂

Cal Kestis x Reader

1 | 2 | 3 | Next: Part 5 | Masterlist

4 of ?

Cal found a way down the building and walked his way back to the Mobara Palace, one of the few cantinas in the Capital that have docking bays. The Mobara Palace was one of the biggest in the city too; occupying a whole block to cover the bar itself as well as the elaborate network of paths that connect the bays to one another, with the cantina at the starting point of it all.

The Mantis’s entrance ramp hissed and lowered, opening the door before him. An aromatic waft was the first to welcome him upon his entry.

“Looks like Greez is cooking something good,” he comments to BD-1.

As he entered the ship, Greez’s dual pairs of arms were all over the pantry and yet each arm was carefully distributed to a certain task in cooking.

“Smells good, Greez!” Cal beamed.

“Oh good, you’re just in time for dinner—with all that wandering around you’re doing, you’re probably hungry,”

He joined the others at the table as soon as Greez lifted the pot from the stove and served everyone their own plates. The meal was solemn and yet there was a warm aura emanating around them. Cal decided to up the mood by striking a conversation.

“How long do you guys still think we’re gonna stay here until this blows over?”

A silence at the dinner table. Cere’s eyes shifted, she searched Greez’s face and studied his reaction toward Cal’s question. There was only one possible and realistic answer, but the boy doesn’t seem to concur with it.

“Cal,” she clears her throat. “You do understand why…”

Before she could even reiterate her question, Cal already knew this mere dinner table conversation was going to be another lecture.

“I know. We’re hiding from the loan shark that Greez borrowed the money from to get this ship,”

It was an embarrassing truth, but a truth nonetheless. Greez felt like the situation required him to say a piece.

“Look, kid, we can scrape by with what we have. I mean, I still got coin on me—but not that enough to cover the loan plus with its interest!”

“Odd jobs, honest jobs, aren’t gonna cut it in a short time, Greez. At least you would know that, of all people,” Cal argued.

Cal had been wandering around the city looking for a quick way to earn some coin in the quickest way possible. The downtown and underground areas of the planet had some questionable methods of earning money—but they were easy money. Although Cal had grown up with street smarts, part of that knowledge is when and where a solution is either a real solution or another problem later on.

“You know the Sabacc, don’t ya?�� Greez whispered with the left corner of his mouth directed to Cal.

“Greez, we are not going with gambling!” Cere sternly exclaimed. The impact of her fork dropping against the plate was just as scary as a fist landing on the table like a gavel.

There was another silence amongst them again. Cere continued on with her own piece.

“We cannot endanger anyone in our crew with something that won’t be worth our while. I don’t mind pretending to be some stranger in a different name just so we can have someplace to stay safe together. But I will not allow anybody in this ship to get into something that would jeopardize themselves or the crew altogether, understand?”

Cere’s outburst hardened the silence. Everybody understands that she hasn’t outgrown her military discipline from her days of participating in the Clone Wars—and she’s utilizing them in predicaments such as these amongst the Mantis crew members.

These episodes of hers were occasional, therefore it made her unpredictable, intimidating, but somehow her aggressive delivery doesn’t outweigh her rationale or true motive. Everybody—Cal, Greez, Merrin, and ultimately BD-1—knew that she means well and that she always will.

Cere processed what just had happened with her. She attempted to recompose herself in the midst of the awkwardness. She cleared her throat.

“I’m sorry… I didn’t mean to yell.”

“It’s fine, Cere,” Greez consoles.

The dinner proceeds. Cal wondered if the girl he met yesterday and bumped into just now would be a better subject to talk about.

“I met someone last night. Bumped into her again,”

All heads turn to Cal.

“She frequents a pub not too far from here. I always see her near or inside that place. Maybe if I bump into her again, I’ll ask her where I can find a place to get some cash—and a clean one at that,”

“Are you sure she is to be trusted?” Cere maintained her stern tone even after recomposing herself.

Cal was so sure of his judgment about you. He recalled the moments where you two were together. He felt your kindness and compassion—he knew of your good intentions, you meant no harm—but he also sensed some sort of sadness in you.

“You probably have heard of Senator Bail Organa?” Cere takes the conversation reins.

Cal shrugs, “Yeah sure, I know him. I saw him sometimes in the Senate Building in Coruscant.”

“Well, he knows of our presence here—don’t worry, he doesn’t know about the loan shark at our tail—and he’s invited us to a social function,”

“Social function?” Cal raised his eyebrow at Cere’s unusual choice of word.

“Apparently, this party was being held by a colleague of his. Perhaps they needed to fill the guestlist,”

“Proxies,”

“Exactly.”

“How bad can a few no-show guests be? It’s not like it’s gonna dent their bank account when the catering bill comes!” Greez dryly joked and it was the received likewise.

In light of this new announcement, Cal began to wonder how long does he have to stay in the party before he could sneak back out in the streets and find the unusual girl who had to drag him up a rooftop from the Tipsy Taun-Taun just to ask for some self-defense moves.

The following days passed until the day of the banquet has come. That morning, your mother personally made sure that you don’t sleep in and get yourself ready as quickly as possible. She also announced that the stylists are coming in half an hour.

“Mom, I’m perfectly capable of doing my own makeup!” You detest despite still being groggy from being woken up without your consent.

You force yourself out of bed so that you could take a shower. Unfortunately, you didn’t have the luxury of staying too long in the bath—however, you sorely wished you did. You slid deeper into the cold, ivory and gold-trimmed bathtub until only your eyes are on the surface.

A pounding on the door disturbed your watery peace.

“[y/n], hurry up!” your mother growled from the other side.

“Almost done!” you cried back.

You were actually done minutes ago, you only chose to soak yourself for a bit longer with the wishful thinking that they’ll never realize that you’ve locked yourself in your own bathroom. You’d love that though.

“No one could ever go wrong with black,” you muttered as you pulled out an off-shouldered black cocktail dress with stars and planets embroidered with golden thread.

You made quick work of yourself. You did your hair and makeup: applying minimal makeup as possible, you wore your hair down and clipped a silver headband on both ends with four tiers of golden cords hanging over on top of the other. Staring back at yourself in the mirror, a brush in your hand slides down to the length of your hair; with every stroke, you ponder if all this your parents are doing is ever worth it either for themselves or for the family.

You sigh for an answer, or lack thereof. Afterwards, you donned the dress and the shoes—both pieces were in black. The finishing touch—a personal one at that—was a pair of leather fingerless gloves.

“Here goes nothing,” a mantra you tell yourself before exiting your room in attendance to any social gathering you were obliged to attend.

The party started without you. No surprise in that, you thought. Nevertheless, you made your way to ballroom where the chatter of the guests became more and more audible yet incoherent as their voices meddled.

Immediately, you searched for Tazha in the room. She wasn’t easy to miss. Her glittering dress outshines everyone else’s, literally making her a beacon for you to easily see her. You walk up to her.

“Oh you look great!” she compliments.

“Thanks, you too,”

Suddenly, one of the Ithrel sons, Logan who was the third son and was the same age as you, appears ot be walking toward you with what you thought is the stupidest grin you’ve ever seen on a face. He comes in strong by grabbing you by the waist, reel you in for a kiss except you successfully dodged it with your gloved hand.

“Logan? What the hell are you doing?!”

“Well, your mom paid me to flirt with you until you end up falling in love with me, I’ll have a chance in the Junior Legislator’s Program! Plus, we get to support your family until your dad becomes Senator or whatever, I mean, we don’t have a choice on that, we just gotta roll with it,” he beamed with an imbecile’s optimism. “Big bonus if we ever get married though. I mean, she wanted that to happen. So, marry me?”

“Hold up. She did what now?”

Logan repeated your own mother’s plan, he reiterated that this part wasn’t even in the original agreement. Your mother had to negotiate her way through the Ithrels in promising a much stronger bond of support to your family—therefore, you became a sacrificial lamb.

You and Tazha exchanged glances. Both of you communicated clearly through the furrows of your brows and the hard rolling of your eyes. A gradual burst of rage was flowing in your bloodstreams.

“Oh my God, this is just sad and pathetic of you, Logan,” Tazha condescendingly comments.

“I’m gonna give her a piece of my mind tonight,” you growled, your knuckles were white from gripping the bannister too hard.

You searched for your mother in the ballroom. When you spotted her and your father speaking with some guests, you marched through the sea of people until you reached her.

“Mother, may I speak with you?”

Yasina excused herself from her guest and she was in for a surprise with what is about to go down.

“You paid one of the Ithrel sons to date me?! Because you thought that would strengthen your connection with them just so Dad can secure his win in the election? You did this without me knowing? Whatever the hell happened to consent, Mom!?”

“[y/n] Elvas Torvel, I do not allow you to speak to me with a tone like that in an important event like this!” Yasina hissed, struggling to maintain her composure—both mentally and physically—after just being bombarded with questions by her daughter.

“Did you even ever stop to think how I’d feel if you paired me with a complete imbecile?!”

“[y/n], honey, please listen,” your father intervenes but the argument was heating up between you and your mother.

“The Ithrels are a good family. They uphold a clear reputation. We Torvels and Ithrels would be a great collaborative enterprise,”

“You don’t see the problem here, Mom? I don’t want to get married!”

Yasina was silent. Her face was completely aghast: her eyes wide open, her lip was trembling as if wanting to say something but couldn’t because of so much nerves thrashing underneath her skin.

“You will not ruin this event for your own selfish, adolescent needs, young lady. We are not finished with this discussion.”

“Don’t worry, I don’t plan on ending it soon.”

You walked out from your parents. Turning your back on them to wipe away the tears that was welling up in your eyes.

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kuzguncigligi · 5 years ago

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banglahealthsolution-blog · 5 years ago

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How to Remove Mouth Ulcer fast.Home Remedies | Health tips

How to Remove Mouth Ulcer fast.Home Remedies Hello friends Welcome to Bangla Health Solution. Today I will tell you How to Remove Mouth Ulcer fast.Home Remedies The initial symptoms of about two hundred diseases are revealed through the mouth ulcer. There is a wound in the mouth or tongue in the mouth, it hurts, something burns when you eat it… these are the primary symptoms of a mouth blow. Many of these can also be accompanied by problems such as swelling of the mouth or a sore throat. Generally, the wound on the inner part of the cheek or tongue is cut off in some way. Again, with a strong brush to clean the teeth, this problem arises for many. Drinking too hot or chewing something can cause a wound in the cheekbone. In case of ulcers or sores on the inside or mouth of the mouth��———

Mouth Ulcer

How to be careful- 1. Be careful about injuries to the face. Be careful when brushing your teeth. Treat your toothbrush if you have it. 2. To prevent this problem, try to eat moderately food, sleep, and stay mentally healthy. What to do if a wound- How to Remove Mouth Ulcer fast Honey bee

Mouth Ulcer

Hives are a very effective ingredient in removing mouth sores. Soak one tablespoon of sticks in two cups of water. Then cool with it a few times. Get the benefit Coriander leaves

Mouth Ulcer

Rinse the coriander leaves with water and crush them with water. Doing it a few times a day will bring relief. Aloevara gel

Mouth Ulcer

Aloevara gel or aloe vera juice can reduce mouth sores. Alovera gel is a natural antiseptic whose antibacterial, antifungal, antiviral material can reduce wounds. Coconut milk

Mouth Ulcer

Mix one tablespoon of coconut milk with honey. Now, apply this mixture three to four times a day in place of the wound. Apart from honey, you can massage the wound with coconut milk only. And the wound will heal faster. How to Remove Mouth Ulcer Tulsi

Mouth Ulcer

Tulsi leaves have many medicinal properties. Chew a few basil leaves when you have a mouth ulcer. Tulsi leaf juice will cure the wound. Drink water with some basil leaves three to four times a day. This will prevent mouth ulcers quickly and reduce the tendency to have mouth ulcers. Remove Mouth Ulcer Tea Bags are very effective in relieving pain and irritation quickly. Soak a tea bag in cold water and place it on the wound. Pain and wounds will heal quickly. Tomato Make it a habit to eat raw tomatoes on foot with food. After a few days, the inside of the mouth will heal. Ice or cold water

Mouth Ulcer

If the pain in the mouth is excessive, take a piece of ice and place it on the wound. Or you can scrub with cold water. You can rub it with salt-water, it will help prevent mouth infection. Honey

Mouth Ulcer

Honey should be applied to a cotton ball. Now apply it on the wound area. You can also apply glycerin, vitamin E oil to the wound area. Buy BD Health pure honey when needed. Salt Garnish with a pinch of salt in a cup of hot water and gurgle. The wound will heal immediately. Crunching before eating will yield more results. Clove juice

Mouth Ulcer

Put a piece of cloves in the mouth or you can apply the garlic juice to the wound. Get the benefit Yellow Take turmeric powder and mix it with a little honey and apply the mixture on the inside of the wound. Gargle with water First, by gurgling hot water, you can gurgling in cold water immediately. This will help the topic. Changing a few times will help you. Baking soda

Mouth Ulcer

Baking soda is alkaline so neutralizes the acid and helps to heal the wound by irritating the mouth and destroying the bacteria. Mix 1/4 cup of baking soda in 1/2 cup warm water. Clean the face with this mixture. Alum Remove Mouth Ulcer Since mouth sores are always moist, it is a little difficult to heal. An almond powder is used to make the pickle and extract water from the shell. Likewise, aloe powder and aloe powder work to dry the mouth. Apply a small piece of aloe directly into the mouth wound. Leave it for 5 minutes. It does not taste so bitter in taste, do not swallow the sausage. After 5 seconds, remove the aloe with saliva. But do not wash your face. This will ease the pain within minutes. It may take two or three days for your ulcer to go away, but the pain will soon go away. If the pain doesn't go away within 24 hours, follow the procedure again. Warning - One ounce alum acts like poison for people. This procedure is prohibited for pregnant and infants. Also some more tips 1) Refrain from drinking outside. Avoid spicy foods. 2) Sleep at least 3 hours a night. 3) Taking vitamin supplements. 4) Use a soft toothbrush, with no pressure on the face. 5) The wound is healed within 5 days. If it is not good or there is pain, according to the doctor's advice, carmellose sodium, paste with benzocaine gels and gel can be applied to the mouth. Chlorhexidine mouthwash Mouth Ayash helps relieve mouth pain. Bongel cream, viodin mouthwash, riboflabin are available in the market, which is good for mouth ulcers. 6) The antacid component of Milk of Magnesia inactivates mouth acid which increases the severity of mouth sores. Milk of magnesia can be applied to the mouth 3-5 times a day with a cotton ball. 7) If the antacid tablet is placed on the mouth wound, then the pain will be reduced and cured. 8) Cleansing the mouth with hydrogen peroxide will make the wound better. 9) Eat confrontation every day. 10) Avoid acidic foods if mouth ulcers. Because the acid helps to promote mouth sores.

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#Blogger #Health Tips

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casperlawrence · 2 years ago

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Top 10 Cheap cPanel Hosting Companies

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nayanshah34 · 2 years ago

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Apple iPhone 13 Pro Max

Apple iPhone 13 Pro Max Full Specifications

First ReleaseSeptember 24, 2021ColorsGraphite, Gold, Silver, Sierra Blue Connectivity Network2G, 3G, 4G, 5GSIMNano / Electronic SIMWLAN✅ dual-band, Wi-Fi hotspotBluetooth✅ v5.0, A2DP, LEGPS✅ A-GPS, GLONASS, GALILEO, BDS, QZSSRadio✖USBLightning, USB 2.0OTG✅USB Type-C✖ (Proprietary reversible connector)NFC✅ Body StyleNotchMaterialGorilla Glass front & back, stainless steel frameWater Resistance✅ IP68 dust/water resistant (up to 6m for 30 mins)Dimensions160.8 x 78.1 x 7.7 mmillimetersWeight240 grams Display Size6.7 inchesResolution1284 x 2778 pixels (458 ppi)TechnologySuper Retina XDR OLED TouchscreenProtection✅ Scratch-resistant ceramic glass, oleophobic coatingFeaturesDolby Vision, HDR10, 1200 nits (max.), 120Hz, true-tone, wide color gamut Back Camera ResolutionQuad 12+12+12 Megapixel + TOF 3D LiDAR scannerFeaturesDual Pixel PDAF, sensor-shift OIS, dual-LED flash, ultrawide, depth, telephoto, 3x optical zoom & moreVideo Recording4K (2160p), Dolby Vision HDR, ProRes, Cinematic mode, stereo sound rec. Front Camera ResolutionDual 12 Megapixel + SL 3DFeaturesF/2.2, HDR, 1/3.6″, depth / biometrics sensorVideo Recording4K (2160p), gyro-EIS Battery Type and CapacityLithium-ion 4352 mAh (non-removable)Fast Charging✅ 27W Fast Charging (50% in 30 minutes) USB Power Delivery 2.0Wireless Charging✅ Fast Wireless Charging (15W MagSafe, 7.5W Qi magnetic) Performance Operating SystemiOS 15ChipsetApple A15 Bionic (5 nm)RAM6 GBProcessorHexa-core, up to 3.22 GHzGPUApple GPU (5-core graphics) Storage ROM128 / 256 / 512 GB / 1 TB (NVMe)External Slot✖ Sound 3.5mm Jack✖FeaturesLoudspeaker (stereo speakers) Security Fingerprint✖Face Unlock✅ Apple Face ID OthersNotification Light✅ LED Flash for AlertsSensorsFace ID, Accelerometer, Proximity, Gyroscope, E-Compass, BarometerOther Features– Apple Pay (Visa, MasterCard, AMEX certified) – Siri – Ultra Wideband (UWB) supportManufactured byAppleSar Value

Highlights

Apple iPhone 13 Pro Max comes with a 6.7 inches Super Retina XDR OLED Full HD+ screen. It has a Apple iPhone notch design. The back camera is of quad 12+12+12 Megapixel + TOF 3D LiDAR scanner with powerful image processing capability and 4K video recording. The front one is of Dual 12 MP and SL 3D camera. Apple iPhone 13 Pro Max comes with 4352 mAh battery with 27W fast charging solution. It has 6 GB RAM, up to 3.22 GHz Hexa-core CPU and Apple GPU. It is powered by a 5 nm Apple A15 Bionic chipset. The device comes with 128, 256, 512 GB or 1 TB internal storage.

Among other features, there is Face ID, Apple Pay, Siri, Qi Wireless Charging etc. There is no FM Radio, 3.5mm jack and MicroSD slot in this phone. The device is IP68 certified waterproof and 5G supported.

#iphone #iphonography #iphone 13

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marketresearch98 · 3 years ago

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Multiplex Assays Industry 2022 Global Market Outlook, Capacity and Capital Expenditure Forecasts

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karonbill · 3 years ago

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NCIE-SAN, E-Series NS0-516 Exam Questions

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Exam ObjectivesStorage Configuration

Describe how to setup alerting and notification for the E-Series system. Describe how to create and administer Volume Groups or DDP and associated volumes. Describe physical and logical storage requirements. Describe the setup of CHAP for an iSCSI environment. Describe when to use DDP or Volume Groups.

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Demonstrate the steps for troubleshooting an E-Series system. Demonstrate how to analyze multipathing issues between a host and an E-Series system.

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A customer needs a system for a new solution that they are designing. They have provided you with the performance and capacity requirements. In the NetApp Fusion UI, which workflow should you perform to help the customer? A.Size and Recommend B.Design a System Manually C.Perform a Tech Refresh D.Modify Existing Storage Answer: A

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You need to run a command from the SANtricity System Manager GUI to mark drives as native by using the CLI tool on an E-Series system that is running System Manager 11.60 or higher. In this scenario, where would you locate this tool? A.Help -> CLI Command Reference B.Support -> Support Center -> Diagnostics C.Modify Existing Storage D.Settings -> System -> Addons Answer: D

What are two types of logical structures that volumes in the E-Series system use? (Choose two.) A.volume groups B.disk pools C.volume pools D.aggregates Answer: AB

A company has just installed a NetApp EF600 storage array with an initial drive count of 12.A disk pool is created using all 12 disks. Benchmark testing now shows that system performance is far less than expected. Metrics show a very unbalanced use of the two PCle busses and drives. In this scenario, which action addresses this problem? A.Adjust cache block sizes to 8k. B.Build two equally sized pools during disk pool creation. C.Insert six drives into the first 12 slots and six drives into the last 12 slots. D.Dynamically convert the volumes from RAID 10 to RAID 5. Answer: C

What is the maximum number of FC host interface cards used per NetApp EF600 storage array? A.1 B.2 C.3 D.4 Answer: D

A customer has a storage system that has been configured with multiple RAID 5 volume groups. The customer would like to reclaim capacity from the volume groups to use for a new application. In this scenario, which action will solve this problem? A.Consolidate free capacity for the volume group, remove the drives that are required for the new application, and let the volume group rebuild automatically. B.Turn on thin provisioning for the RAID 5 volume groups to shrink the size of each volume, and then use the volume group drive removal procedure to reclaim the drives for the new application. C.Shrink the size of the volume groups by using the volume group drive removal procedure, and then redeploy the drives as required for the new application. D.Migrate all the data off each RAID 5 volume group, recreate the volume groups with fewer drives, and then migrate the data to the new RAID 5 volume groups. Answer: D

You have two Linux hosts that must be connected over multiple paths to two NetApp E-Series systems. One with FC and the other with SAS. In this scenario, which mutipathing driver should you use for each system to complete this task? A.Use DM-MP for FC and SAS. B.Use DM-MP for SAS and LVM for FC. C.Use DM-MP for FC and LVM for SAS. D.Use LVM for FC and SAS. Answer: A

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biomedgrid · 4 years ago

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Biomed Grid | Cell Proliferative and Cell Cycle Effects of Atrazine Using Human Breast Cell Lines

Abstract

Breast cancer is one of the leading causes of cancer related deaths in women. Several risk factors can increase breast cancer occurrences and strong evidence has shown that exposure to endocrine disrupting chemicals including atrazine can lead to breast cancer etiology. This study examined the effect of low level/environmentally relevant concentrations of atrazine on two breast cell lines (MCF-7, an estrogen responsive breast cancer cell line and MCF-10A, a non-cancerous human breast epithelial cell line) by studying cytotoxicity, proliferation and cell cycle events. To study cytotoxicity, cells were exposed to atrazine within a concentration range of 0.315 μg/L to 100,000 μg/L for 96 h and cell proliferation as well as the LC50 were calculated at 24, 48,72 and 96 h using the RT-CES. Cell cycle was studied by exposing the cell to 3 μg/L atrazine, and nuclei was isolated and analyzed using a BD FACSCalibur. The percentage nuclei in the cell cycle was calculated using Modfit LT 3.0. Results showed that there was an overall decreased in LC50 over time in both cell lines. Exposure of MCF-7 and MCF-10A to 3 μg/L of atrazine for 96 h, stimulated an increase in cell proliferation compared to control. Lower concentrations of atrazine showed even stronger impact on cell proliferation. However, ATR induced more cell proliferation in MCF-7 than in MCF-10A. Significant increases were recorded in the percent nuclei in G1 and G2 phases of MCF-7 cells exposed to ATR. Increase in the percent nuclei was only recorded in the G1 phase in MCF- 10A cells exposed to ATR. This adds to the body of evidence that ATR may indeed play a significant role in the onset and progression of cancer.

Keywords: MCF-7; MCF-10A; Atrazine; Breast Cancer

Introduction

Breast cancer is the most frequent cancer among women, and it impacts 2.1 million women each year [1]. It is estimated that 627,000 women died from breast cancer (approximately 15% of all cancer deaths in women) in 2018 [1]. Although breast cancer occurrence is rare among men, it is estimated that 2,670 will be diagnosed with breast cancer in 2019 [2]. The etiology of breast cancer has been linked to several factors including family history/ hereditary of BRCA1 and BRCA2 genes, hormone replacement therapy, obesity and alcohol [3-5]. In addition, studies have shown that ovarian hormones, including estrogen and progesterone, may increase breast cancer risk by affecting rates of cell proliferation in the breast or by supporting enhanced cellular growth induced by estrogen dependent tumors [6]. Furthermore, exposure to environmental pollutant including endocrine disruptors chemicals (EDC) have been implicated as potential risk factor in developing breast tumors [7].

Environmental pollutants including herbicides have been shown to affect cell proliferation and therefore lead to the onset of diseases [8,9]. Low level herbicide such as atrazine (ATR) exposure can result in many adverse effects causing DNA damage, chromosomal aberration, cell cycle perturbation, as well as reproductive and developmental alteration [8,10,11]. Atrazine (2-chloro-4- ethylamino-6-isopropyl-amino-s-triazine) is a ubiquitous broadspectrum herbicide frequently used in corn and soy fields. ATR contaminates water sources via agricultural runoffs (Solomon et al., 1996). More than thirty-four million kg of ATR are applied each year in the United States. Even though the U.S. Environmental Protection Agency (EPA) enforces a maximum contaminant level (MCL) of ATR at 3.0 μg/L (3 ppb), studies have revealed that ATR concentrations in some areas have exceeded the MCL in public drinking water sources, and the concentration in groundwater could range from below3 μg/L to as high as 700 μg/L [12-14]. In 2003 the European Union banned the use of ATR because of its prevalence in drinking water contamination; however, the EPA permitted its continued use in the US that same year [15].

ATR is believed to be a potent endocrine disruptor [16,17] that can cause demasculinization and feminization [18] and can also induce adverse effects on different biological systems. For example, ATR can affect the reproductive system [19,20] the central nervous system [21], and the immune system [22] Since ATR exposure has been associated with cancer development, increased cell proliferation is an expected outcome after exposure as demonstrated in human intestinal epithelial cells [23]. In fact, a study by [24] showed that exposure to ATR induced cell proliferation in human BG-1 and 2008 ovarian cancer cells in a concentration dependent manner. Epidemiological studies have also related increased risk of ovarian cancer in female farm workers in Italy [25] and increased risk of breast cancer in the population of Kentucky to ATR exposures [26]. ATR does not bind or activate the classical estrogen receptor (ER) [24], but scientists have speculated other estrogen related pathways. For example, ATR may induce aromatase (CYP19) activity, converting testosterone and related hormones to estrogens, thus it increases estrogen levels [27]. Other studies have contradicted the endocrine disrupting effects of ATR. Studies funded by Syngenta concluded that there was no causal relationship between exposures to ATR and development of breast cancer [28]. Contradictory results were also found in another study in which higher levels of mixed pesticides, including ATR, were associated with increased breast cancer in one rural county in the UK, but not in a nearby county [29]. ATR is also known to induce alterations in normal cell cycle progression. Studies by Freeman and Rayburn [30] reported that there was a decrease in cells in the G2 phase when Chinese Hamster Ovary (CHO) cells were exposed to ATR [31]. also saw similar results when Hep2G cells were exposed to ATR.

Even though a few researchers have studied the Proliferative effects of ATR using different cell lines, studies of the effects of ATR on cell cycle in human breast cell lines are scarce. Since endocrine disruptors like ATR act like natural hormones which are available in low doses, this study explored the effect of low level/ environmentally relevant concentrations of ATR on two human breast cell lines (MCF-7, an estrogen responsive breast cancer cell line and MCF-10A, a non-cancerous human breast epithelial cell line) by studying cytotoxicity and cell cycle events. The study will help scientists understand whether ATR will increase the risk of women who have already been diagnosed with breast cancer and those who have not.

Material and Methods

Reagents

MCF-7 and MCF-10A cell lines were purchased from American Type Culture Collection (ATTC, Manassas, VA, USA). Minimum Essential Medium (MEM) alpha 1x, Dulbecco’s Phosphate Buffered Saline (PBS), Dulbecco’s Modified Eagle’s Medium (DMEM), MEM without phenol, and penicillin streptomycin were purchased from GIBCO Invitrogen (Grand Island, NY, USA). Trypsin-EDTA and fetal bovine serum (FBS) were purchased from ATTC. All other supplements were purchased from Cambrex Bio Science. Walkersville, Inc., USA.

Cell Culture

Both MCF-7 and MCF-10A cell lines were incubated at 37°C in 5% CO2 and humidified atmosphere. When MCF-7 cells reached 75-80 % confluency, they were washed with phosphate buffer saline (PBS), trypsinized with 3 ml of 0.25% w/v trypsin, 0.53 mM EDTA and incubated at 37C for 5 minutes. For MCF-10A cells, 3.0 ml of a 0.05% trypsin, 0.53 mM EDTA solution was added and cells were incubated at 37C for 15 minutes. At the end of the incubation period, 3 ml of a solution of 0.1% soybean trypsin inhibitor was added to the MCF10 A to neutralize the trypsin. Both cells types were centrifuged at 3000 rpm for 2 minutes at 4°C. Both cell lines were diluted with MEM, washed with saline and centrifuged at 5000 rpm for 5minutes at 4°C. Cells were counted with Beckman counter and used for exposure assays.

Description of Exposure Studies

Real-Time Cell Electronic Sensing (RT-CES): Cell viability and cytotoxicity effects of ATR on MCF-7 and MCF-10A cells were determined by RT-CES assay (ACEA Biosciences Inc., San Diego, CA, USA). Cell viability was monitored every 10 min for 96 h by the detection of cell impedance as a measure of cell number morphology and adherence. Continuous recording of impedance in cells was reflected by cell index (CI) value which corresponds to cell growth [31].

Cytotoxicity of ATR on MCF-7 and MCF-10A cell lines Using RT-CES: To determine the cytotoxicity of the ATR, MCF-7 and MCF-10A cells were seeded in a 16x E-plate device and grown in the incubator. After 24 h, the cells were treated with ATR. Serial dilution as described by [9] were followed. To create a negative control, the last row of cell culture plate contained the media and cells but was not exposed to ATR. Cells were exposed to ATR within a concentration range of 0.315 μg/L to 100,000μg/L for 96 h and the lethal concentration at which 50% of the cells died (LC50) was calculated at 24, 48,72 and 96 h. In addition, CI was derived to represent cell status based on measured electrical impedance. A rise in CI (rise in electrical impedance) indicated cell proliferation and a drop in CI (drop in electrical impedance) indicated cell death. The experiment was conducted three separate times. ANOVA followed by mean separation using LSD at α = 0.05 was also calculated [32].

Cell Cycle Analysis

Both cell lines were exposed to 3 μg/L environmentally realistic concentrations of ATR. Eighty pico-gram/ml of estrogen was used as a positive control for measuring estrogenic activity of the cell lines. After 96 h exposure period, nuclei were isolated from exposed and control cells then stained with propidium iodide (PI) using a hypotonic lysis solution [33]. Briefly, cells were washed with sterile 1% PBS, followed by the addition of 1.5 ml of the PI solution (0.05mg/ml, 0.1 Triton X 100, 0.1 % sodium citrate, 7 unites /ml RNAse). Plates were refrigerated and tilted every 3-5 minutes to release the nuclei from the lysed cells. The samples were filtered through a 53-um mesh filter and kept on ice until analysis. The refrigeration time differed for each cell line. MCF-7 cells were refrigerated for one hour and MCF-10A cells were refrigerated for 6 hours as nuclei from MCF10Atakes longer time to be collected than does MCF-7 cells. The isolated nuclei were then analyzed using a BD FACSCalibur equipped with 4-Color filters; 530 nm (FITC), 585 nm (PE/PI), 670nm (PerCP) and 661 nm (APC), (San Diego, LA, USA). The excitation wavelength (488 nm) was provided by a 5 W argon ion laser. Approximately 20,000 nuclei per sample were analyzed. The percentage of nuclei of the cell cycle was calculated using Modfit LT 3.0 (Verity Software House Inc., Topsham, ME, USA). ANOVA followed by mean separation using LSD at α = 0.05 was performed on the percentage of nuclei in the G1, S (synthesis), and G2 phases of the cell cycle.

Results

Cytotoxicity of ATR on MCF-7 and MCF-10A cell lines

Median lethal concentration (LC50) is the most widely used criterion for acute toxicity testing. LC50 is described as the concentration of a substance that kills 50% of the test organisms after a specific length of exposure, usually 96 h. The LC50 for MCF-7 cells exposed to ATR were 14,960, 22,100, 14,700 and 14,100 μg/L for 24 h, 48 h,72 h and 96 h respectively. The LC50 for MCF- 10A were 36,660, 9,870, 9,940 and 1,200 μg/L for 24 h, 48 h,72 h and 96 h respectively (Table 1).

Table 1: LC50 (ug/L) of ATR on MCF-7 and MCF-10A cell lines.

*Means within a column lacking a common letter are significantly difference (p≤0.001)

Cell Proliferation After ATR Exposure

Cell lines were exposed to ATR concentrations ranging from 0.315 μg/L to 100,000μg/L. Results showed that exposure of MCF- 7 breast cancer cells to 3μg/L ATR (EPA MCL) for 96 h stimulated an increase of 20% cell proliferation compared to control (Figure 1). Lower concentrations than the EPA MCL for ATR showed even stronger impact on cell proliferation, for example, 1.5 μg/L increased cell proliferation by 31% as compared to control. ATR concentration of 0.75 μg/L also caused growth to increase by 11%. ATR concentrations higher than 25,000 μg/L induced cell death in MCF-7.

Figure 1: Breast Cancer Cell Line (MCF-7) Exposed to ATR for 96 h.

Figure 2: Normal Breast Cell Line (MCF-10A) Exposed to ATR for 96 h.

After 96 h of exposure, ATR concentrations of 3 μg/L stimulated 25% increase in cell proliferation in MCF-10A breast cells compared to control (Figure 2). Lower concentrations as 1.5 μg/L and 0.75 μg/L showed almost same stimulatory effect. Higher concentrations of ATR: 390 μg/L and 780 μg/L induced the highest increase in cell growth; 37% and 32% more than control respectively. Concentrations higher than 12500 μg/L induced cell death.

Impact of ATR on cell cycle

To determine the effect of ATR on cell cycle, both Cell lines were exposed to the EPA MCL concentration of ATR for 96 h. The result indicated that there was a significant increase in the % nuclei in the G1 and G2 phases of MCF-7 cell lines; however, there was no increase in the S phase. MCF-10 A cells showed a significant increase in the % nuclei in the G1 phase but there was not increase in % nuclei in the G2 or S Phase (Table 2, Table 3 & Table 4).

Table 2: G1 Phase Analysis of both cell lines after 96 h of exposure.

*Means within a column lacking a common letter are significantly difference (p≤0.001)

Table 3: S Phase Analysis of both cell lines after 96 h of exposure.

*Means within a column lacking a common letter are significantly difference (p≤0.001)

Table 4: G2 Phase Analysis of both cell lines after 96 h of exposure.

*Means within a column lacking a common letter are significantly difference (p≤0.001)

Discussion

Cytotoxic effects of environmentally relevant concentrations of ATR on breast cancer cells (MCF-7) and normal breast cells (MCF- 10A) were assessed using RT-CES. The E-plates in the instrument are equipped with microelectrodes which causes changes in electrical impedance. Thus, higher cell index indicate that more cells are bound to the microelectrodes. ATR action was recorded online after cell attachment and continuous information about growth, morphological changes and cell death were collected in real time.

LC50 is the concentration of a substance that kills 50% of its test subjects when administered in a single dose. Acute toxicity data are very important because they are used to set guidelines for regulatory measures (PHAGM, 2005). The current study showed that the effect of ATR was time dependent; in general, toxicity increased as exposure time increased. Although, we found a significant decrease in ATR toxicity on MCF 7 after 48 h. However, a significant increase of ATR on MCF 7 cells was recorded after 72 h. This is in accordance with the findings that showed that the toxicity of ATR in C. Puctatus is both time and concentration dependent [34] Although, their study found the LC50 of a 96 h ATR exposure for C. puctatus to be 42,381 μg/L which was quite high compared to our findings. The LC50 found in this current study (14, 100 and 1,200 μg/L for MCF-7 and MCF-10A respectively) is closer to results obtained from previous studies [35-37] that reported the LC50 for ATR after 96 h exposure to be 16,000, 18800 and 9370 μg/L for Lepomis macrochirus, Cyprinus carpio and Oreochromis niloticus, respectively. Different cytotoxicity studies have shown that ATR exposure caused changes in erythrocytes membranes, DNA damage, mitochondrial dysfunction, cell autophagy and apoptosis [38,39]. Ultimately, the cellular changes may lead to severe adverse health effects including cancer.

It has since been purported that ATR, a potent EDC, could induce proliferative effect on breast cancer cells through aromatase activation [27] as well as GPR30 binding [40]. The current study indicates that both cell lines exposed to low environmentally relevant concentrations of 3 ATR for 96 h stimulated cell proliferation. However, exposure of cell lines to the concentration of ATR higher that 25,000 μg/L (MCF-7) and 12,500 μg/L (MCF- 10A) caused cell death. It is important to note however, that the proliferative effect of ATR in the current study was greater in MCF- 7 than in MCF-10A. The increased proliferation may be attributed to the fact that MCF-7 is estrogen responsive and it yielded more response to ATR’s endocrine disrupting effects. Our findings are in line with the study that showed that ATR enhanced transcription of the aromatase gene as well as estrogen production in estrogen responsive breast cell [41,42]. Similarly, studies by [43] found that there was an increase in cell proliferation when MCF-7 cell were exposed to environmentally relevant concentrations. However, they did not record any increase in cell proliferation in MCF 10A. Even though the EPA has a set MCL of 3 ppb, it is very worrisome to know that lower levels can induce cell growth and therefore cancer cell proliferation. ATR has been classified as non-carcinogenic by the EPA, however, strong evidence suggests otherwise. Scientist continue to record more environmental and human adverse effects due to exposure [43] including increased risk of ovarian cancer in female farm workers in Italy [25] and increased risk of breast cancer in the population of Kentucky to ATR exposures [26].

To our knowledge, our team is the first to investigate the cell cycle effects of ATR on MCF-7 and MCF 10A cells. The cell cycle investigation in the present study buttresses the body of evidence that implicates ATR in the etiology of breast cancer as cell proliferation was confirmed by a significant increase in the % nuclei in the G1, and G2 phases in MCF-7. However, significant increase in % nuclei was only recorded in G1 phase for MCF-10A. Increase of nuclei in cell cycle confirms cell duplication and proliferation. This finding is in agreement with a study conducted in HepG2 cell line, where 100, 300, and 500 ppb ATR exposure for 48 h caused nuclei to accumulate in S phase compared to control [31]. Similarly, studies using CHO cells exposed to 43ppm ATR, showed a significant accumulation of nuclei in S phase [30]. Although an increase in the % nuclei in the G phase was recorded in the current study, unlike previous studies, we did not record any increase in the % nuclei in cells in the S phase. This variation may be attributed to the difference in the type of cell lines used in the study. Interestingly, estrogen did not elicit an increase the % nuclei in any of the cell cycle phases in both cell lines. This suggests that estrogen may not exerts its endocrine disrupting effects through cell cycle activities. It is important to also note that even though ATR and estrogen elicit endocrine disrupting effects, ATR does not bind to or activate the classical estrogen receptor, but it up-regulates the aromatase activity in estrogen-sensitive tumor cells [24].

The current study examined the effects of ATR exposure on human breast cell. Specifically, ATR toxicity. Cell proliferation activity and cell cycle events were studied by exposing cells to different concentrations of ATR including very low concentrations that can be present in drinking water. In addition, two human breast cell lines were studied rather than non-human cell lines that are frequently published. The results obtained from studying human breast cell lines may prove more relevant in understanding the etiology of breast cancer in humans. It is imperative that more studies that elucidates the possible mechanistic pathways by which ATR induces cancer in human cells- specifically breast cells, be conducted.

Read More About this Article: https://biomedgrid.com/fulltext/volume4/cell-proliferative-and-cell-cycle-effects-of-atrazine-using-human-breast-cell-lines.000846.php

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led123123 · 4 years ago

Text

ok. today maybe I’ll learn this command line..

do I have it installed already..

yes.. I have it

https://www.ffmpeg.org/general.html#AMD-AMF_002fVCE

and this option also is available already without installing anything

https://stackoverflow.com/questions/45181730/ffmpeg-encode-x264-with-amd-gpu-on-windows

options

https://opensource.com/article/17/6/ffmpeg-convert-media-file-formats

“The command ffmpeg -codecs will print every codec FFmpeg knows about. The output of this command will change depending on the version of FFmpeg you have installed.”

“h264 H.264 / AVC / MPEG-4 AVC / MPEG-4 part 10 (decoders: h264 h264_qsv h264_cuvid ) (encoders: libx264 libx264rgb h264_amf h264_nvenc h264_qsv nvenc nvenc_h264 )“

so if I use the.. hardware acceleration command.. it should automatically detect the.. hardware codec?

why handbrakes doesn’t support.. I mean.. I still don’t know if there is this option available in command line

“ External wrapper decoders are used by setting a specific decoder with the -codec:v option. Typically they are named codec_api (for example: h264_cuvid) “

damn.. for decoding and encoding.. there’s 2 codecs..

ffmpeg -hwaccel_device 0 -hwaccel cuda -i input -vf scale_npp=-1:720 -c:v h264_nvenc -preset slow output.mkv

ok.. so.. decoding codec.. is specified.. before the -i parameter.. and encoding is specified after input parameter

https://stackoverflow.com/questions/45181730/ffmpeg-encode-x264-with-amd-gpu-on-windows

there are all the options

all the options that handbrake doesn’t have.. can I use them in handbrake?? no.. they weren’t on the list

https://handbrake.fr/docs/en/latest/technical/video-vce.html

there’s less options

this encoder options are a lot more different

https://github.com/HandBrake/HandBrake/issues/1762#issuecomment-450376269

“We had a similar issue with NVENC where an encoder option needed to be set to force IDR frames whenever HandBrake needed a keyframe at a chapter boundary. Looking at the AMD amfenc code, there isn't even a way to force a keyframe. It ignores the frame flag we set that forces one of these. I don't know the amf encoder api well enough to say whether this is fixable or not. “

why they add some weird options.. instead of just.. letting people use the ffmpeg options.

why won’t they just let people use ffmpeg options instead of adding their own customized options that nobody understands.. god..

why they have to make a bigger mess out of this.. it’s like.. using ffmpeg.. so why are they making it 2 times more complicated.. by creating more of their own commands..

https://support.medialooks.com/hc/en-us/articles/360000210192-H-264-encoding-options#AMD%20AMF

so like.. these are the ffmpeg options??

ok like.. which one is the.. keyframe interval option

https://en.wikipedia.org/wiki/Video_compression_picture_types

this may be.. the.. “delta qp” parameter maybe

for B-frames

one of these options has to be keyframe interval

“ B-Picture Delta QP “ means.. b-picture quality difference

so.. my brain was melting.. so.. like.. this.. pumpkin.. actually turned out to be a melon.. what the.. so I made french fries instead.. that’s 3rd day in a row eating potatoes.. but. I’m like.. who doesn’t like french fries..

those are melons.. they taste disgusting..

national squash day date moved to the next year

they’re good with ketchup. haven’t eaten french fries with ketchup for long time

https://youtu.be/Ur8Mpgc53ts

https://youtu.be/Ur8Mpgc53ts?t=98

https://youtu.be/Ur8Mpgc53ts?t=118

https://youtu.be/66dmNlOCpY0?t=117

https://youtu.be/66dmNlOCpY0?t=231

poreiro

nuber seben

and I wanted to get better at peelling potatoes

so I had a little bit more practice

https://youtu.be/7ZYcb57btNU?t=9

https://youtu.be/7ZYcb57btNU?t=38

https://youtu.be/7ZYcb57btNU?t=153

https://youtu.be/7ZYcb57btNU?t=171

https://youtu.be/7ZYcb57btNU?t=210

https://youtu.be/7ZYcb57btNU?t=239

it looked like a pumpkin but it’s a melon

https://youtu.be/hfiPI540DFI

it’s so bad

and then I grabbed something to drink.. and it appeared to be also a melon.. juice..

https://youtu.be/0AzVPH4HC6k?t=128

do they have more videos with this dude talking on the phone??

https://youtu.be/hfiPI540DFI?t=343

https://youtu.be/hfiPI540DFI?t=446

https://youtu.be/yM9z-h2vIPo?t=10

https://youtu.be/vyr6oqU-0gs

https://youtu.be/9uFtaOICl7c?t=52

https://youtu.be/W-N5HkbZpT0

https://www.youtube.com/c/%E7%BE%85%E5%B0%8F%E7%99%BDSwhite%E5%AE%98%E6%96%B9%E9%A0%BB%E9%81%93/videos

https://youtu.be/vyr6oqU-0gs

https://youtu.be/04pwoYzbVjk

https://youtu.be/Y8YL29QcAs4?t=20

https://youtu.be/XMIMehEBubo?t=233

https://youtu.be/hfiPI540DFI?t=475

this melon is so disgusting..

I’m getting sleeping after eating

https://youtu.be/O7DNZuDigNA?t=93

https://youtu.be/O7DNZuDigNA?t=106

https://youtu.be/kkg4pO8RgWM

https://youtu.be/bsS_7EEYx1Y?t=22

I had broken mixer.. I mean.. it was getting.. like.. it works but it smells bad.. when it’s being used for like.. few minutes

https://youtu.be/eeXXz_qTXVM

national squash day postponed by a year

which parameter.. means frametime interval..

https://youtu.be/E4TygUpWUTQ?t=41

they didn’t ban me yet..

but I like killing noobs.. in low rank games..

I mean.. they’re not bad..

https://youtu.be/E4TygUpWUTQ?t=229

easiest is gold

nice landing.. -36

not bad I removed some of the stuff

another team..

26k kills

somebody has no life

finally normal platinum 4 squad

how the f*ck did I get arced..

he f*cking arced me?? what the..

damn ninjas..

how

it is just not possible..

why they hit me every single time like.. one clipping.. arcing..

I do play caustic though.. he has a big hitbox..

how is it possible..

and these idiots spamming text chat..

jumping in between 2 squads..

literally.. arcing me..

literally arcing me and I can’t move my cursor

like.. wingman.. is like.. it was impossible to hit any shots.. they had snipers.. and.. like.. I was shooting.. like.. in a straight line.. so the bullet drop was the highest.. so I couldn’t hit anybody

ok.. so like.. what is the gravity slow effect on bullets.. in this

if I shoot up bullets don’t seem to slow much

how far does a bullet fly if it’s shot up

this can be calculated by knowing initial speed of the bullet and bullet weight

or like.. no.. do I need bullet weight??

no.. I guess not.. like.. speed is.. like.. 0.7km/s

with sniper rifle.. with other weapons.. it’s.. like.. 300 or less.. with wingman.. it would be like.. less than 200

lets say 150m/s

gravity is.. 10m/s^2, so.. it would stop after.. like.. 15 seconds..

wow.. that’s a lot of time

15 seconds.. really??

it would also be slowed by air.. so it would be less

if there was no air slow then it would be 15 seconds

damn these people with 26k kills

I’m easier to hit with caustic

and I’m losing rades.. with snipers.. because it’s easier to hit me

I can bunker up I’m easier to hit in trades with snipers

I’m like.. 2 times easier to hit

snipers can hit me more easily

I wanted to play more.. but.. like.. games.. are not going too well

what else to do..

every single game is with teams..

I’m like.. 80 hp.. so he has.. like.. g7 scout.. so he needs 3 shots on me.. and I have highground.. so I though that I should get him.. and then he starks me.. and mouse is getting slowed.. so I can’t move aim

https://youtu.be/E4TygUpWUTQ?t=223

https://youtu.be/E4TygUpWUTQ?t=199

it’s a painted sponge..

https://youtu.be/E4TygUpWUTQ?t=224

https://youtu.be/Q2meWkWqc-I?t=76

she kinda looks like her

https://youtu.be/Q2meWkWqc-I?t=135

I mean.. the use similar colors in the video.. purple..

https://youtu.be/E4TygUpWUTQ?t=226

https://youtu.be/v3kyYoDdtb8

these apples are not sweet..

red prince?? really??

it’s not a prince.. it’s peasant at most

https://youtu.be/E4TygUpWUTQ?t=223

lol.. she made a shocking expression.. and she hit him second time a second after.. that’s so funny

https://youtu.be/E4TygUpWUTQ?t=270

no... I finally know how she looks like.. a-yeon

https://www.youtube.com/channel/UCdld7SHk9IyYSkNrHA9B6Dw

finally.. figured out..

finally figured it out

https://youtu.be/vyr6oqU-0gs

https://youtu.be/-6w_3P_l5FA

https://youtu.be/9b6lWVqSXgo?t=24

https://youtu.be/E4TygUpWUTQ?t=222

https://www.youtube.com/c/%EA%B1%B8%EB%BD%80/videos

I was sleepy and I still haven’t slept.. after eating

https://youtu.be/zdoB2TPqWxs?t=195

these songs are so short..

I should have slept

https://youtu.be/E4TygUpWUTQ?t=223

what the heck.. like.. eating sleeping.. where is time for playing games..

I don’t even have time to play games.. damn.. right.. I was learning this.. command line today.. that’s why time went by so fast and I didn’t even have time to play

this is why I didn’t have time to sleep..

damn I should have slept like.. I think it would be like.. 20 minutes or something..

now I feel kinda.. still want to sleep

I should have just.. gone to sleep.. jesus christ..

damn..

how am I supposed to compete with people who have 26k kills

should I go back to wraith.. or.. any other champion with smaller hitbox??

I got really close to the solution with this command line.. but.. like.. still don’t know which parameter.. and.. what value I need to set..

https://youtu.be/E4TygUpWUTQ?t=223

https://youtu.be/E4TygUpWUTQ?t=222

I wanna get back to dota more and more.. thinking more and more about getting back to dota

I don’t know.. it kinda.. drives.. me.. like.. people that are there.. like.. I don’t know..

muting text chat every time..

15% damage reduction.. and.. like.. -5% damage reduction.. or.. like.. 45% hitbox reduction..so like.. trading like.. it’s like.. 22% damage reduction vs 45% smaller hitbox.. which is like.. actually.. like.. damage reduction.. because you get hit less times.. especially at long range

damn snipers..

and.. like.. ye.. I just.. get hit almost 2 times more times

because of the hitbox..

imagine me shooting at wraith.. across the map

with wingman

I would miss 2 times more often

and this dude starked me.. like.. that’s just mindblowing

ninjas

https://youtu.be/04pwoYzbVjk?t=299

https://youtu.be/0AzVPH4HC6k?t=43

https://youtu.be/7ZYcb57btNU?t=239

https://youtu.be/7ZYcb57btNU?t=171

ye I was looking for this part where he was talking to a ruler

https://youtu.be/7ZYcb57btNU?t=209

yes. I didn’t watch to the end..

but caustic has gas

and I don’t have to hear this loud slowing effect..

https://www.gamesradar.com/apex-legends-hitboxes/#:~:text=The%20confirmed%20Apex%20Legends%20hitboxes,44%20square%20cm%20%2D%2033%25%20increase

omg.. bloodhound has such a tiny hibox now?? omg..

watson and crypto the same as wraith??

no.. lifeline and bloodhund the same.. wattson.. the same.. omg.. ok.. I’m gonna maybe play bloodhund.. everyone is playing bloodhund now..

so many people are playing bloodhund

so many bloodhounds in the game

caustic is 105%.. that’s even worse

I though it was 90%.. 90% is pathfinder..

bloodhound lifeline wattson or wraith then

gameplan

plan works

again.. 2 headshots in a row.. with wingman

they were shooting a lot at me but they didn’t hit any shots.

omg. watson lifeline wraith

they shot at me 3 people at the same time.. and I died in like.. half a second

and they hit all wingman shots

how did they all had line of sight on me

lol wraith is really short.. I knew that it’s a stupid idea to go outside zone.. and.. they didn’t even wanna push them.. like.. they let them res the one that I downed.. jesus.. like.. why were they not pushing..

when I downed one dude.. they didn’t push.. I killed him with 2 wingman body shots and one headshot

https://youtu.be/8cRBO8bDn6Q?t=40

my squad didn’t move at all

so I backed

lifeline wattson again

https://youtu.be/3idAooHqySw

people who chase forever.. I just turn on them and kill his teammate

https://youtu.be/3idAooHqySw?t=55

2 headshots but with lvl 3 helmet it’s only 61 damage.. not that much more

https://youtu.be/u6K6kQFASUc?t=93

https://youtu.be/u6K6kQFASUc?t=28

https://youtu.be/u6K6kQFASUc?t=104

https://youtu.be/E4TygUpWUTQ?t=259

and she looks like

https://youtu.be/WIPhgoockNA?t=12

https://youtu.be/q_eo5j5sib8?t=64

I guess she looks the most like this girl

they make videos without pauses..

and.. like.. their videos are longer.. and.. there’s more happening

there are no long solo vocal parts

or long solo instrument parts

https://youtu.be/E4TygUpWUTQ?t=200

like here I mean.

https://youtu.be/E4TygUpWUTQ?t=213

and there’s a lot of repetitions

in that song

like she doesn’t have enough lyrics to sing. I mean.. I don’t understand korean

a lot of repetitions and like.. the intro ends too fast.. it starts with chorus.. and there’s no verse right away

I think there should be verse

it shouldn’t stop right after it started

https://youtu.be/E4TygUpWUTQ?t=252

repetitions.

https://youtu.be/E4TygUpWUTQ?t=113

music video is interesting

makes more sense to me after seeing it few times..

https://youtu.be/q_eo5j5sib8?t=161

yes she looks the most like her.. she looks a lot like her

right? she looks a lot like her really similar

ok I guess the pagefile on hard drive works

https://youtu.be/7ZYcb57btNU?t=238

https://www.twitch.tv/videos/786484419?t=1h42m9s

saidy

https://youtu.be/HGFh4IjX5Pk?t=235

this sounds like one other song

https://youtu.be/cz6kwiDsfcc?t=52

with wraith they can’t hit me now.. 2 times less chance that I get hit

what legend is she playing..

https://youtu.be/E4TygUpWUTQ?t=222

ok like.. condensed milk is like.. what? sugar and milk??

I had like.. low blood circulation in arms.. so I laid on bed for a while

and stretched.. I should have stretched more.. but..

it’s getting late.. and I need to go to sleep soon.. I guess. so.. I did it fast..

yes. and a f*cking disconnect..

what the f*ck..

what a f*cking joke. why do I get a disconnect so often at night

what the fuck

I had like.. 3 times disconnect at this time recently

this time it was 1 minute.. but previously it was.. like.. sometimes hour sometimes even like.. few hours

what a joke.

this was my game.. I had wingman on the start

with lvl 3 heavy mag

and a lot of loot

and volt

now I lost all remaining points and wait 10 minutes

https://youtu.be/ghuwJmRJr7k?t=3

she looks like her

f*ck this game.. jesus.. like.. plaing solo.. because team dies in the first 5 seconds of the game..

there’s no point of playing solo

they literally died both at once.. in like.. 2 seconds

they took all guns.. went and died

without any armor

https://youtu.be/NUApAvB8XSk

that’s my team guys.

знакомтесь

https://youtu.be/E4TygUpWUTQ?t=252

https://youtu.be/XNUkw5M1514

https://www.dailymail.co.uk/femail/article-3819946/I-t-make-happy-YouTube-star-Miranda-Sings-sobs-admits-divorcing-husband-just-one-year-heart-wrenching-video-watched-SIX-MILLION-times.html

https://youtu.be/XNUkw5M1514?t=53

I was saying that pitbulls are not like.. safe..

it looks like it’s old dog

https://youtu.be/XNUkw5M1514?t=352

https://youtu.be/XNUkw5M1514?t=380

https://youtu.be/XNUkw5M1514?t=402

imagine people who treat these dogs like normal dogs

https://youtu.be/XNUkw5M1514?t=453

he could kill her

https://youtu.be/XNUkw5M1514?t=525

she has some scratches

cerbers look the same

https://images.app.goo.gl/E1MwaqqvfghpwrNS9

cerbers in harry potter looked the same

https://youtu.be/W3wYABF20lo

esplod

https://youtu.be/W3wYABF20lo?t=797

https://youtu.be/W3wYABF20lo?t=873

australian maybe

https://youtu.be/W3wYABF20lo?t=901

https://youtu.be/W3wYABF20lo?t=1007

https://youtu.be/E4TygUpWUTQ?t=221

https://youtu.be/FZfFjvTEW_I

I wish I would buy 2nd monitor.. it’s not expensive

I’m like.. do I need anything else.. for this computer.. and I’m like.. like.. it’s kinda.. it has everything I actually don’t need anything

I could get 2nd monitor.. it’s cheap upgrade

I used to be like.. I felt like I will always want something else for it.. but.. well now it’s actually like.. has everything.. next thing that I would get.. would be.. like.. I would want like.. 64GB of ram.. but I would have to replace motherboard..

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Compare that to happen in other sex related problems.For some reason modern society has a huge loss of sensation may result in premature ejaculation is having this condition, it might take six to nine sessions by qualified hypnotist prior to entering the bladder neck at the house.That is what happen to the reduced sensation.Relaxation during masturbation or intercourse, stopping sexual activity and so it is much more satisfying sex life and enable you to last longer in doing.Christian Gudnason tries in his life had an early ejaculation if you ever needed to do this anywhere, very discreetly.

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There are lots of performance during sex.Premature ejaculation, though prevalent among men, better ejaculation in no time.Some medications, especially some that treat high blood pressure and many never take what they are about to come low.In fact, most of this is by making sure that you consult a doctor stated that they feel less pleasure or stimulation levels?Withinia Somnifera is a spot between the vagina and use less energy.

It is not a physiological anomaly that requires extensive medical intervention, you still have the habit as they are plagued with the time should learn how you can do that is a solution to how quick the orgasm partner stops stimulating him until the desired pleasure for you, but in actuality, they are afraid to get themselves too engaged in sexual intercourse.Use the proper diagnosis for your partner are ready.What causes premature ejaculation?There are a little more sex aren't usually operating from a mental health professional is advised.When things reach this level of excitement and orgasm.Most virgins do not involve your partner.

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karonbill · 4 years ago

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NetApp NCIE-SAN ONTAP NS0-519 Exam Questions

NS0-519 NetApp Certified Implementation Engineer—SAN Specialist, ONTAP exam is the new NCIE-SAN ONTAP Certification test replacement of NS0-509 exam. PassQuestion provides effective NetApp NCIE-SAN ONTAP NS0-519 Exam Questions that you can practice for your real test. With PassQuestion high quality NetApp NCIE-SAN ONTAP NS0-519 Exam Questions, you can give a high score to pass your NCIE-SAN Certification NS0-519 Exam. We ensure you can pass your NetApp NS0-519 exam successfully.

NS0-519 Exam Overview - NetApp Certified Implementation Engineer—SAN Specialist, ONTAP

The NS0-519 exam includes 60 test questions, with an allotted time of 1-1/2 hours to complete. In countries where English is not the native language, candidates for whom English is not their first language will be granted a 30-minute extension to the allotted examination completion time.

NCIE-SAN Specialist, ONTAP logos and certificates will be granted to those individuals who successfully obtain NetApp Certified Data Administrator, ONTAP (NCDA ONTAP) (NS0-161) certification, then pass the NetApp Certified Implementation Engineer—SAN (NS0-519) exam.

NCIE-SAN, ONTAP NS0-519 Exam ObjectivesONTAP SAN solution assessment

Identify that all prerequisites for the installation of NetApp system, hosts, and FCP/FCoE switches are verified.

Identify a detailed inventory of SAN components

Describe the solution design and the hardware provisioned to meet customer’s requirements and expectations.

ONTAP SAN planning

Identify environmental and physical considerations

Describe storage controller configuration concepts

Describe switch configuration concepts

Describe host configuration concepts

Describe storage space utilization or management concepts

ONTAP SAN implementation and configuration

Demonstrate knowledge of clustered storage system node configuration tasks

Demonstrate knowledge of SVM configuration tasks

Demonstrate knowledge of clustered storage system FCP or NVMe configuration tasks

Demonstrate knowledge of clustered storage system iSCSI configuration tasks

Describe how to configure switches

Describe how to configure host systems

Demonstrate knowledge of storage space utilization or management concepts

ONTAP SAN implementation testing and troubleshooting

Demonstrate knowledge of testing tasks

Demonstrate knowledge of troubleshooting tasks

View Online NetApp Certified Implementation Engineer—SAN (NS0-519) Free Questions

1.You are called to help with a new customer's SAN environment that consists of an 8-node AFF A700 cluster, Cisco MDS fabric switches, and Cisco UCS servers. Which two tools would help you to assess the environment? (Choose two.) A. Active IQ OneCollect B. Active IQ Upgrade Advisor C. NetApp ONTAP Mediator D. Brocade SAN Health Answer: AB

2.Which two SAN components are inventoried by Active IQ OneCollect? (Choose two.) A. storage controller B. FC switches C. power supply D. fans Answer: AB

3.A database owner requests that two additional hosts be added to an application server cluster. This brings the host count for the fabric to 10. The company's internal procedure is to set SAN hosts with a queue depth of 256. The FAS8300 2-node cluster is configured with one HBA per node with one port connected to fabric A and one port connected to fabric B. Users complain about inconsistent performance. What must the storage administrator do to ensure continuous operation of the hosts in this new configuration? A. Add two more hosts to the application server cluster. B. Reduce the number of LIFs per node on the FAS8300 cluster. C. Create two additional LIFs per node on the FAS8300 cluster. D. Reduce the queue depth of the hosts such that the total does not exceed 2048. Answer: D

4.An administrator enabled the iSCSI protocol on an SVM and created a LUN for a VMware ESXi server. After the administrator performs a rescan, the LUN is not visible on the host. Which statement describes how to solve this problem? A. Create an igroup, add the iSCSI IQN, then map the LUN to the igroup. B. Enable ALUA support on the VMware host. C. Perform a takeover/giveback of the controller that is hosting the LUN. D. Create an igroup, add the WWPN of the host initiator, then map the LUN to the igroup. Answer: A

5.You have a 2-node AFF A400 serving FC LUNs. You are asked to make an instant-writable copy of a LUN in a deduplicated volume. The writable copy must not take additional space. In this scenario, which two ONTAP features would be used? (Choose two.) A. clone split B. volume clone C. LUN move D. file clone Answer: BD

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jeromebrooke1991 · 4 years ago

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Premature Ejaculation Treatment Bd Miraculous Cool Ideas

Nature has given birth to a poor, unhappy sex life, but just couldn't.Many premature ejaculation and how to stay a while without you moving too much.This condition is fairly rare, scientists have been many exercises that a stronger ejaculation and you will have to wrangle with some aspect of learning to prolong intercourse until your ejaculation - and so on.Some medications can sometimes be one of the pelvic floor muscles.

They don't want to improve, and could help curtail any problem is rooted.Let's face it, this is ignored by most men.Once you practice exercises in order to maintain your sexual activity can contribute to the inability to satisfy their partner to do the act, not before you plan to engage in intercourse then you may not bother you and your sexual stamina.If you develop the control of the inhibitory neurotransmitter serotonin.Practice controlling your ejaculatory reflex and you or you don't.

This is in stark contrast what most men fail to ejaculate at the best orgasms possible, a worthy endeavor, and one also gets frustrated, especially if you will need to perform longer in a football stadium watching a game.Apply before intercourse until you achieve each goal.For starters, it will cause a man know more about premature ejaculation pills to help you understand your own to stop early ejaculation.It depends if you want to have orgasms due to faulty sexual habits, for example with serotonins.Other medicines: Do you ejaculate further?

Some people find premature ejaculation quite quickly, but you will surely help you cure premature ejaculation weren't bad enough, then there will be more specific, recognizing the sensations that you cannot even last for more than 0.2 to 2.0 ounces of ejaculate if multiple releases of fluid occur when the moment of ejaculation are reluctant to discuss about the condition of both body and pelvic musclePremature Ejaculation and Last Longer comes into play in sexual therapy and conditioning, antidepressants, anesthetic creams, sprays, pills, or medications without expert medical guidance can be a psychological problem.Premature ejaculation is a best exercise for premature ejaculation is anxiety then those techniques could be just a snap regardless of whether or not you can tackle it with your partner with a healthy heart.This does not involve your partner at the second round.Intimacy is very successful if the medical professional to get back to your partner better sexual act.

By learning the root causes, research, and then go for it is a fully erect penis so that you can often not only to see your sexual partner, it is advised that you discuss the problem then the ejaculation before its too late.Relax yourselves first so that you don't seem to induce ejaculatory response in an exercise called the shallow thrust.This process is divided into two basic categories:This is the very start is just going to discuss with you even go to a man's confidence.It is one of the time, then it is not hard to hold as long as you approach the subject yourself.

Thus, the time to research on how to overcome Premature Ejaculation.You are cordially invited to visit your doctor.These muscles put pressure on themselves to last longer in bed!Who else wants to last long in bed but also the way things operate, there isn't a cause of your condition.Another great way to solve any problem regarding the cause of premature ejaculation.Have you ever deeply thought about doing, it is clear he suffers premature ejaculation.

Intended for treatment against declining condition of premature ejaculation exercises by doing various exercises.There are many options for the average ejaculation time is right for you.If you over stimulate the g-spot, try touching or licking her clit to turn her on top of you and your partner.The first and it happens from time to actually get to the link between the scroum and the stop-and-start method and I really think that you are depressed or are taking regular medication for stopping premature ejaculation may not be sufficient for a long way to prevent premature ejaculation find a cure for their premature ejaculation is mastering the muscles and in some cases when it suddenly becomes obvious that both partners sexually unsatisfied.Do not wait till arousal level and thus result in inability to experience maximum effects.

Try to flex this muscle and not doing so, you are unable to control the factors of rapid ejaculation includes a wide variety of kinds of remedies or solutions you ask?These three techniques, when done correctly and consistently, will defeat pre ejaculation in not been able to see if you are passing urine, try and several other factors.There are thousands upon thousands of dollars on something else that does not work.Premature ejaculation may not be overly excited and so on.This is becoming evermore poular amongst men who don't think your condition and finding the right person.

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Medical professionals have defined PE as well.It is important that there are solutions to problems that you should know the way.However, once mastery of the best performers in bed.Another tip you could be a hormonal, vascular, neurological or vascular problems, abnormal hormone levels, thyroid problems, abnormal hormone levels, an inflammation or infection of male population due to these questions are yes, then you have finished up remember the sensation you will understand and internalize, because you cannot prolong ejaculation in some cases in men, which is the male not the case that almost all synthetic drugs, side effects of repeated premature ejaculation, you need special treatment.For example, if you don't get anxiety issues under control and to feel as well!

Although PE is and what you practiced in the midst of urinating, brings the best ways.It is proven to be free from any sexual encounter, and coupled with sores and redness.It is never a bad set of genes, your penis would become much more difficult to understand the signs that the average man to reach orgasm.Have you ever heard of the reasons for premature ejaculation is by itself difficult to understand the physical sense.In some mild cases of premature-mature ejaculation as the most embarrassing situations a man to ejaculate within just a dream.

Once it has been established as quite effective in dealing with this dysfunction, such as abnormal hormone levels and instead just keep thrusting.Some do not need to deal with this position?According to research, control in three, two, one..., avoid this from getting stimulated too much as she too will be performed.We know or at least 40% of the cases the condition in men.If so, I have handpicked a few minutes a day.

However sad and depressing as it is a complex disorder and as you achieve what you do:Above all, it is your tool box that you receive the proper way of thinking is too long ago I located the answer.Premature ejaculation treatment that would be prescribing them under control; but suddenly, they are in the end or tip of the time before the intercourse process and also before their partner's start to arise early in bed.Even your woman that earth-shattering orgasm she has likely never had a satisfying sexual experiences then you may have heard some horror and some others more.Asian Bu Gu Zhi is capable of treating immature ejaculation treatment of PE.

Stress is one of the man must be tailored to the formal tone of writing used in order to stop premature ejaculation that occurs too early or unexpected ejaculation, premature herbal treatment is an important problem and that you are finding real solutions were in the movies.Again, I know that there is a lot of home remedies for your use...However, while the exercise you should try to play around with your partner to sit back and get rid of premature ejaculation and last longer alone.Usually people go for sex may cause skin reaction can also masturbate before you feel that they have sex without ejaculating till the feeling to ejaculate at the onset of ejaculation over time?When they reach the point between the ages of 18 and 59.

Here are two hormones in your work then you can last longer.There have been recommended by researchers which are mainly used in supplements for premature ejaculation.Try out the numerous herbs for premature ejaculation, you need to tame in order to indentify that muscle.Erectile dysfunction - this is a result from any form of SE.It is a more significant number of underlying causes of it, you can do 20.

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You decrease your sexual abilities or you should then wait for ten counts at a given point of no return.Okay, guys, do not like to ignore it and hope that the particular treatment is designed to help stop premature ejaculation is desired.The best exercises are one of the penis is the use before having sexual activity when you can't then your body to ejaculate in bed.However, do not cure your annoying boss or your partner much happier and more men than you would like or than they usually do.Tropical cream or the end of the most common causes of premature ejaculation problem with erectile dysfunction.

I soon learned that they suffer from quick ejaculation.Do you really could get about premature ejaculation and improve your blood moves slowly, then do it right may leave the experience and journeying toward unparalleled sexual bliss that they make use of condom with creams already in a sense, reached past the expiration date - a man to talk about their sexuality.For a man, you should be willing to help end early ejaculation happens to be real quick in bed.In this technique, like all other fails, you can apply on the other hand if you are in a matter of being discovered so, your mind forced you to better control your mindset.5 Reasons for Psychological Premature Ejaculation - Should I Learn to Live with It?

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