#cv frisk

[cvbullshit]

The urge to make cat versions of CV characters is real

[haveyouseenthisskeleton]

Day 9 with a broken inbox, still no news but stay calm. Here goes some Edge headcanons today (Underfell Papyrus)!

Edge has a cat problem on the Surface. A big cat problem. It's not his fault. Everytime he sees an abandoned kitten, all he can think is how growing outside all alone is hard because he lived it and he takes it home. The only problem being that after some months, there are cats everywhere and his house smells kinda bad. So Edge did some research and learned how to be a good foster dad. He's now taking three cats at the same time (+ his old girl Doomfanger) and helps them get better until they are ready to find a new home. It's better that way, even if he can't stop himself from death threatening every new owner if they dare to give up on his babies again. That's why the rescue organisation prefers to be there when he's giving away the cats now. Edge only keeps the very old and very ill cats who don't have long to live so they at least have some nice house while they need it. He loves being litters' mom too. He has a soft spot for orphans kitten.

Despite his impressive CV, Edge struggles to find and keep jobs on the surface. Since he has no use as a royal guard anymore, he's not sure what to do with himself. He tried a lot of things, from being a florist to being a body guard, but something is not working, everytime, and he either quits or gets fired because that doesn't match. Edge wonders if something is wrong with him or if maybe he is just not made to work, but that's for sure hurt every time. He hopes he can find some stability soon, and something he really likes, but for now, he keeps moving from jobs to jobs.

Red and Edge are the only pair of brothers who don't live together anymore. After they reach the Surface, they decided for their own good to live in different appartments. It's not because they hate each other, on the contrary, they're closer than ever before, but they are bad at just... living together, only screaming and fighting all the time. They prefer to have their own spaces, and things work a lot better between them since.

Edge doesn't have a car licence, but he thinks the examiner is wrong and drives a car anyway. Clearly, the man simply didn't understand his genius when, during his exam and to avoid a traffic jam, Edge sped on the guardrail and flies above the cars waiting to jump across the river and shortcut through the forest to join the road again. What should he had done? Wait like all these peasants?! Clearly that man was wrong! He made his own car licence. Police officers didn't appreciate it when they arrested him not even a year after.

He accidentally food poisoned Frisk's entire school with his broken glass lasagnas. Then Toriel came and kicked his butt until he accepts, on his knees, to recieve cooking lessons from her. He still thinks it's humiliating but god, everyone is thankful his cooking is finally improving, and especially Red who's eating at his house every sunday. Red cries the first time it was edible and teleported to Toriel's place to kiss her full mouth. Toriel slapped him so hard he almost fell down.

[atomograd]

hi! this is my sideblog :3 cutting for length, not for any bad/content warning related reason

a lot of stores do sell canes, crutches, braces, and walkers. medical supply stores sometimes sell off the rack wheelchairs, and there are many wheelchairs online - but all of these generic aids are about as useful as the reading glasses at target for people who need them for more than the basic on-the-box descriptions. often these aids can be harmful in other ways, such as causing pressure sores, tendonosis, misalignment, or crushing injuries. i had this experience when my insurance refused to cover fittings for back braces and suffered injury from one off the rack, and it was actually cheaper for me to buy custom medical corsets from established professionals than have just the fitting for a back brace, let alone purchase it afterwards. [we also see this happen with many trans care related products, baby products, menstruation products, adult diapers especially ones designed for people with external genitalia, translation services, et cetera...]

the use and need between a pair of glasses is actually hardly different from a wheelchair. the more you need the aid, the more specialised it will have to be. the more you need it, the harder it is to access the aid with the specifications you need. the less you have it, the more disabled you become.

one of the major things making so many people think differently of prescription lenses VS wheelchairs for example, is... cost. glasses tend to be less expensive than wheelchairs, so insurance covers them more and more people can buy them. glasses are also less expensive to accommodate, but they aren't always despite this. we have to remove glasses when we step through a metal detector just like we must stand from our chair or be frisked, for example

if you can't walk without hurting yourself because you can't see... i don't see that as too different from how i can't walk without being injured and needing to not walk even more. subtitles, braille, and other visual access features are not standard in places like medical facilities - how many times has a provider noticed you wear glasses and ask if you need a braille version of standard new patient paperwork...? most providers don't offer that at all, but many low vision people with glasses need braille. and there are high quality screenreaders and similar devices that could make that paperwork more accessible...! but it's difficult to access, heavily censored with few speakable words for reproductive health and other language, and doesn't always help someone know where to find the lines to write their responses on. mobile devices help with this but again, they are the CVS reading glasses or rite-aid knee brace or costco hearing aid version of this aid.

i'm not disagreeing with you now that you've further elaborated! i wanted to add some thoughts i didn't want to spam your reply section with. ultimately the most important thing for people who use aids, or want/need to, to focus on is unity. we should all dedicate the time spent on competing in the disability olympics to taking care of each. other, networking, and improving access - and now we know how to get in touch with each other to do just that! i know a lot about navigating insurance and finding alternative solutions for care and aids, if you or someone you know needs help or resources. thanks for chatting!

"Having to wear glasses/contacts is a disability"

Me, having worn glasses since idk kindergarten: No....

[cri-spy]

Tumblr artists UwU

Camila Cuevas/@camilaart

Jael /@jakei95

@digital-template

@justsleepyskelly

@polilla-de

@cloudthesmallone

Bonus!

Billy Bob/@the-quiet-kid-dark

[coziroses]

Day 7: Suspicious

Day 8: Wish

Curse asf.

Curse as fuck 2

Hello welcome back. My bad for not posting yesterday I did it at nighttime and i was to lazy and tired to post it, Plus there is some curse drawing that someone requested Blueprint and i drew Eternal to be part of this. Because why not.

Blueprint belongs to @pepper-mint . Sorry if it is curse asf

Kano, Mílo , and Eternal belongs to me

Earth frisk and star belongs to @spacetale-offical

The fourth one is a funny joke with theses two weirdos @damn-bro-you-ugly and @yo-it-deylight

[asktheapsquad]

what would each member of the aps squad do if kiwi lost their voice for whatever reason?

Kiwi: well actually I did lose my voice once and everyone reacted differently

Kiwi: when I first realized I lost my voice I was a little upset so pizza comforted me!



kiwi: and frisk decided they wanted to show me how to play other instruments Turns out there really good at playing the xylophone 



Kiwi: The doctors I talked to said I should have some medicine so hat kid went and got some for me For some reason she didn’t tell me where she got it from

Kiwi: and Henry… well let’s just say he talked for me… sometimes he would take advantage of that but most of the time he helped me so I wouldn’t strain my voice



Kiwi: still I’m glad everybody cares about me so much :) 

what the first image says:

Kiwi: sad they can’t sing)

Pizza: it’s OK it.won’t be gone forever

what The third image says:

Miriam: why can’t you just buy medicine again?

Hat kid:well I’m banned from CVS…haha…

What the fourth image says:

Henry:they’re trying to say fuck you

[triundertale]

Hi guys! So, we're only two updates away from catching up with the preboot, huh... So let's reboot it again!

This time, I'll make sure that EVERYTHING is EXACTLY like what the fandom wants :)

So, I've killed frisk and gave their soul to Asriel so he can be alive (because that's how it works) and now sans is the protagonist and very sexy sexy tongue man blue eye.

I've also decided to split Chara into two characters: Chara Dreemurr who is very nice and cute and loves everyone, and Chara (said chair-ah) who is very evil mean sex gril and does a murder stab goopy face and is love sans? 😳

I've scrapped asgore bc he boring and i don't know how to draw berd. So gaster is now main charater hes very secsy stoic amine man with hands.

every other character also got a redesign to make them look like all the very 100% canon fanart, and from now on every post will look exactly like this to cut in time so we can update every day :)

Oh, and on a totally completely unrelated note, happy April Fool's day! :D

-CV CL?

[lilli-chae]

Some more CV HorrorFell AU concepts! Sans and Frisk designs not finalized, and Undyne is just a brainstorm sketch for rn - HOWEVER! these are the boys' (and the original fallen human's) canon heights ^^

[lynn-does-stuff]

Ever realize you have way to many OCs

(OC list under the cut)

Anna

Tyler

Kate

Konnor

Molly

Emma

Tony

Russel

Rewind

Legs

Ghost

Lilith

Josie

Daniel

Memory

Kayla

Jaxson

Lilia

Neige

Kimo

Widow

Fans

FDT!Chara

FDT!Frisk

Twitch

Type-R

Fuben

Azmera

Slate

Cotton Candy (CC)

Slice

Dice

CV!Lynn

Parasite

Flynn

Lophi

Rei

Terra

Ryver

Ijema

Cukaro

Nixehek

Afukiy

Pubox

Onone

Surobib

Udah

Apotol

Etelug

Zeletehi

Ibozej

Orep

Vimuyata

There’s probably more too.

[villainousshakespeare]

My husband is a light skinned black man, I am a very pale Anglo Saxon woman. We have dark skinned nephews and a niece who often stay with us. In the ten years we have been together I have witnessed:

1. A white woman calling the cops on him “because I wanted him to go away” (I still think my getting there before the cops was the only reason he didn’t go to jail)

2. Police following him through a subway station talking on a radio about the “suspicious looking guy” until he got to me and gave me a hug, then everything was alright.

3. Him being targeted by stop and frisk

4. Security tailing my nephew and I around CVS while we shopped

5. A bowling alley not wanting to rent shoes to my niece

6. My nephew being pulled over for driving while black and having his car ransacked by drug dogs

7. An officer asking if I “wanted to be there or was under duress” when we were caught makig out in a secluded area

8. My nephew not being allowed to use his credit card at a store because they insisted it must be stolen

This is just a small sample of what they put up with every day. I don’t know how they have not snapped yet.

I am so depressed and angry on their behalf; he is simply exhausted and sad. Please, if you have white privilege, do whatever you can to be an ally. They need all of our voices. This is so beyond okay.

[hostingnewsfeed]

Stocks To Watch: Tencent Music, Starbucks And Under Armour In Focus

New Post has been published on http://rentts.org/stocks-to-watch-tencent-music-starbucks-and-under-armour-in-focus/

Stocks To Watch: Tencent Music, Starbucks And Under Armour In Focus

Welcome to Seeking Alpha’s Stocks to Watch – a preview of key events scheduled for the next week. Follow this account and turn the e-mail alert on to receive this article in your inbox every Saturday morning.

Investors are prepping for another week of volatility as concerns over the China trade deal and the impact of the U.K. Brexit withdrawal bill are sure to linger. Add in the drama of a yield curve that inverted on the short end and it’s small wonder that the Santa Claus rally is having a hard time materializing. With major indexes having now erased their gains for the year, expect to hear some noise from value investors after seeing the one-month drops in names such as Nvidia (NASDAQ:NVDA) -25%, Target (NYSE:TGT) -22%, Tiffany (NYSE:TIF) -22%, Goldman Sachs (NYSE:GS) -19%, Apple (NASDAQ:AAPL) -18%, Halliburton (NYSE:HAL) -15% and Altria (NYSE:MO) -15%. On the economic front this week, new reads on producer prices, consumer prices and retail sales will be closely watched.

Notable earnings reports: It’s a very thin week for earnings reports, but companies due to spill numbers include Ascena Retail (NASDAQ:ASNA) and Stitch Fix (NASDAQ:SFIX) on December 10; American Eagle Outfitters (NYSE:AEO) and Dave & Buster’s Entertainment (NASDAQ:PLAY) on December 11; Phototronics (NASDAQ:PLAB) and Oxford Industries (NYSE:OXM) on December 12; Adobe (NASDAQ:ADBE), Costco (NASDAQ:COST) and Ciena (NASDAQ:CIEN) on December 13.

IPOs expected to price: Tencent Music Entertainment (TME) is pressing ahead with its IPO as the company looks to take advantage of the 90-day pause on tariffs between the U.S. and China. The music streaming service plans to sell 41M ADSs in the offering and selling shareholders will also unload just under 41M ADSs. Tencent Music owns three streaming music platforms in China – QQ Music, Kuguo, and Kuwo – with a count of over 700M monthly active users. Heading into the IPO, Tencent (OTCPK:TCEHY) holds a 58.1% stake in Tencent Music and Spotify (NYSE:SPOT) owned a 9.1% stake.

IPO lockup expirations: Greenpro (OTCQB:GRNQ) on December 10; US Xpress Enterprises (NYSE:USX) and Charah Solutions (NYSE:CHRA) on December 11; Avalara (NYSE:AVLR), Verrica Pharmaceuticals (NASDAQ:VRCA) and Puixin (NYSE:NEW) on December 12.

Analyst quiet period expirations: Vapotherm (NYSE:VAPO) and Eton Pharmaceuticals (NASDAQ:ETON) on December 10; Weidai (NYSE:WEI) on December 11;

Projected dividend changes (quarterly): Abbott (NYSE:ABT) to $0.30 from $0.28, AES (NYSE:AES) to $0.14 from $0.13, Amgen (NASDAQ:AMGN) to $1.39 from $1.32, Boeing (NYSE:BA) to $2.08 from $1.71, Franklin Resources (NYSE:BEN) to $0.25 from $0.23, Lilly (NYSE:LLY) to $0.57 from $0.5625, Realty Income (NYSE:O) to $0.221 from $0.2205, AT&T (NYSE:T) to $0.51 from $0.50, Ventas (NYSE:VTR) to $0.81 from $0.79, ABM Industries (NYSE:ABM) to $0.18 from $0.175, Balchem (NASDAQ:BCPC) to $0.46 from $0.42, SEI (NASDAQ:SEIC) to $0.32 from $0.30, Urstadt Biddle (NYSE:UBA) to $0.275 from $0.27, WD-40 (NASDAQ:WDFC) to $0.59 from $0.54.

60 Minutes: Tesla (NASDAQ:TSLA) CEO Elon Musk was interviewed by Leslie Stahl for the weekly news show. In a clip already released, Musk said “I don’t really want to try to adhere to some CEO template.” Musk also stated that the EV automaker would have interest in setting up shop in an old General Motors (NYSE:GM) plant if one is available.

Analyst/investor meetings: Arthur Gallagher (NYSE:AJG), Enbridge (NYSE:ENB), American Water Works (NYSE:AWK), ServiceMaster Global (NASDAQ:SERV), TreeHouse Foods (NYSE:THS) and Crown Holdings (NYSE:CCK) on December 11; Aimmune Therapeutics (NASDAQ:AIMT) and Hess (NYSE:HES) on December 12; ImmunoGen (NASDAQ:IMGN), Health Insurance (NASDAQ:HIIQ) and Danaher (NYSE:DHR) on December 13; Hartford Financial (NYSE:HIG) and Centene (NYSE:CNC) on December 14.

Spotlight on coffee: Starbucks (NASDAQ:SBUX) hosts its biannual investor day event on December 13. The meeting will occur about a month after Starbucks announced a round of corporate-level job cuts. While the company isn’t directly in the line of fire of tariffs, an escalation of the U.S.-China trade battle could end up hurting its business if Beijing slows the regulatory process or a consumer backlash pops up.

Spotlight on Under Armour: Under Armour (UA, UAA) will hold an investor meeting on December 12. CEO Kevin Plank, President, COO Patrik Frisk, CFO David Bergman and other Under Armour execs will provide an overview of the company’s long-term strategy, financial outlook and key initiatives to deliver sustainable, profitable growth and shareholder value.

Capitol Hill watch: Google (GOOG, GOOGL) CEO Sundar Pichai is due to appear before a House panel on December 11 in a hearing rescheduled from last week. Height Capital Markets expects the tough questions to be asked to Pichai by lawmakers to be just the start, not the end. The firm thinks Big Tech companies will be exposed to regulatory headline risk into 2019, even as it predicts final legislation will be tough to pass both houses. The House is also slated to vote next week on a bi-partisan measure that would punish drugmakers trying to game Medicaid’s rebate system to garner bigger profits.

FCC watch: The FCC is due to start its quadrennial review of some media ownership rules, including the issue of owning multiple stations in a single market. Companies watching the developments include Entercom (NYSE:ETM), Emmis Communications (NASDAQ:EMMS), Cumulus (NASDAQ:CMLS), Cox Radio (CXR), Clear Channel, CBS, Disney (NYSE:DIS), Beasley Broadcasting (NASDAQ:BBGI), iHeartMedia (OTCPK:IHRTQ).

Short report: Nasdaq is due to issue a new short interest report on December 11 for positions settled by November 30. Stocks with the highest level of short interest as a percentage of total float at the time of the last Nasdaq report included GNC Holdings (NYSE:GNC), Lannett Company (NYSEMKT:LCI), J.C. Penney (NYSE:JCP), Carbo Ceramics (NYSE:CRR), Carvana (NYSE:CVNA), Buckle (NYSE:BKE), Revlon (NYSE:REV), RH(NYSE:RH), Dillard’s (NYSE:DDS), Camping World (NYSE:CWH), B&G Foods (NYSE:BGS) and Pyxus International (NYSE:PYX).

M&A tidbits: Lumentum’s (NASDAQ:LITE) acquisition of Oclaro (NASDAQ:OCLR) is expected to close on December 10. Shareholders with Akebia Therapeutics (NASDAQ:AKBA) and Keryx Biopharmaceuticals (NASDAQ:KERX) vote on the merger between the two companies on December 11. A special meeting is scheduled on December 11 for shareholders of Dell Technologies (NYSE:DVMT) to vote on the Class V common stock exchange transaction. CVS Health (NYSE:CVS) has a deadline of December 14 to issue a briefing defending the Aetna (NYSE:AET) deal.

Sales updates: Franklin Resources (BEN) on December 10, TD Ameritrade (NASDAQ:AMTD) on December 11; E*Trade Financial (NASDAQ:ETFC) and Charles Schwab (NYSE:SCHW) on December 14.

More Prime Time: It’s another notch on the belt for Amazon (NASDAQ:AMZN) as its streaming service launches on Comcast’s (NASDAQ:CMCSA) Xfinity X1 platform.

Cyberwatch: Zix (NASDAQ:ZIXI), Calix (NYSE:CALX) and GigCapital (NYSE:GIG.U) are scheduled for one-on-one meetings at the Cowen 5th Annual Networking & Cybersecurity Summit on December 12.

Barron’s mentions: The share price drop on Caterpillar (NYSE:CAT) looks overdone, reasons Jack Hough. He notes that a midyear price increase by Caterpillar on machines helped offset higher costs related to steel and other tariffs, while booked orders are strong for 2019. Applied Materials (NASDAQ:AMAT) is also seen as being on the cheap side, with shares trading at only 10X forward earnings. That’s despite the huge upside for Applied Materials in AI chips. There’s also a deep dive on Lam Research (NASDAQ:LRCX) after the company’s bumpy road.

Sources: Nasdaq, EDGAR, Reuters, CNBC, Bloomberg

Editor’s Note: This article covers one or more microcap stocks. Please be aware of the risks associated with these stocks.

[cvbullshit]

CV Frisk

[biomedgrid]

Biomed Grid | The Nucleocapsid Protein Gene as Excellent target for Detection of Canine Distemper Virus by Reverse Transcriptase-Polymerase Chain Reaction

Introduction

The Canine Distemper Virus (CDV) belongs to the genus Morbillivirus of the Paramixoviridae family, Mononegavirales order and is a pleomorphic virus with a lipid envelope, a diameter of 150 to 300 nm, whose genome is a single-stranded RNA, with a negative sense and with a helical nucleocapsid.

The genome consists of about 15.7 kilobases (kb), which codes for six structural proteins: the nucleocapsid protein (N), the phosphoprotein (P), the matrix protein (M), the polymerase (L) and the envelope glycoproteins, the fusion protein (F) and the haemagglutinin (H). These last two, located in the lipid envelope surrounding the virion, are responsible for the recognition and entry of the virus into the host cell [1-4]. The helical nucleocapsid contains the N, P and L proteins, which initiate intracellular replication. The M protein connects the glycoproteins of the surface and nucleocapsid during viral maturation [1,5].

The N gene, of around 1.5 kb, is one of the most conserved genes of the CDV genome and it has highly conserved regions in the first 2/3 of the gene between members of the genus Morbillivirus and encodes the most abundant of the structural viral proteins, the nucleocapsid protein, which has regulatory functions of transcription and replication, as well as the encapsidation of the RNA genome in a resistant RNAsa nucleocapsid. However, despite being a highly conserved region of the CDV genome, some variations of the N gene have been shown among field isolates [6-9].

Diagnosis of CDV. Of the diagnostic methods, the clinical signology is the main tool used by veterinarians, however, the non-specificity of the signs in some cases makes it impossible to reach a final diagnosis, so it is necessary to use laboratory tests. Among them, one of the most used is the ELISA (Enzyme-Linked Immune Sorbent Assay), which can detect serum antibodies (Abs) IgM (against the nucleocapsid proteins, N and P of the CDV) or IgG (against the antigens of the envelope, H and F of the CDV). However, this test does not differentiate if the Abs produced correspond to maternal Abs, vaccine or infection. Otherwise, the production of Abs depends on the stage of the disease and may not be in the initial and final phases of it. In dogs not vaccinated with acute infection, they can die without presenting Abs against the virus. In animals with neurological signs, the determination of specific Abs against CDV in cerebrospinal fluid not contaminated with blood, is a definitive diagnosis of DC encephalitis, however this is a very invasive method and its results are variable [7,10].

Another diagnostic method used is the detection of eosinophilic intracytoplasmic inclusion bodies by cytology or by immunofluorescence, however, these can only be visualized in certain periods of the disease, generally being absent when clinical signs are generated [10,11]. Immunohistochemistry is used to detect viral antigens and/or inclusion bodies in tissues, with reliable results only when there is a marked viremia, and this technique can be performed exclusively on samples taken postmortem [7,9]. The isolation of the virus has a very high sensitivity and specificity, however it is a laborious and very slow procedure, so it is not routinely used for diagnosis [11]. The RT-PCR consists of an exponential amplification of DNA fragments of the virus, after reverse transcription from RNA to complementary DNA, allowing the presence of the virus RNA to be detected early, being a Unlike the other techniques mentioned, a sensitive, specific and rapid test for the diagnosis of CDV can be positive even when other tests fail to detect the virus [7,10,11]. In the last decades, several methods based on RT-PCR for the diagnosis of CDV have been developed, being the main targets for amplification, genomic regions that present a high degree of conservation among the virus isolates. This is how, as in 1999, Frisk et al. developed a method, based on the amplification by RT-PCR of a conserved 287 bp fragment of the N gene, which was later used by several authors, proving to be a very effective technique for the detection of the virus genome [7,8,12]. The main objective of this title memory is to diagnose CDV molecularly, through the implementation of the RT-PCR assay of the nucleocapsid protein gene.

Materials-and-Methods

This work was carried out in the Virology and Microbiology laboratories of the Department of Animal Preventive Medicine of the Faculty of Veterinary and Animal Sciences of the University of Chile, with financing from FIV PROJECT 121014019102010

Controls

Positive controls: For positive vaccinal controls, blood was used from the negative control without vaccination, contaminated with live attenuated CDV virus obtained from the vaccines “Nobivac® Puppy DP” (Onderstepoort strain), “Canigen MHA2PPi/L” (Lederle strain) and “Vanguard” Plus 5/CV-L “(Snyder Hill strain). For this, each vaccine was reconstituted with 0.5 mL of 0.9% sodium chloride, and then this mixture was added to 0.5 mL of blood, to later extract the RNA. The ten positive controls from national isolates of the virus corresponded to viral RNA. These isolates came from domestic dogs from Santiago de Chile and were confirmed as positive by a previous RT-PCR based on the hemagglutinin gene [13,14]. Negative controls: Blood was used with anticoagulant of two uninfected animals, of an adult dog without clinical signs of the disease, without a risk and without vaccination history and of an adult dog without clinical signs of the disease and without a risk history, but with a calendar of daily vaccination (vaccinated for the last time ten months ago).

Suspicious samples of DC

Eleven samples of peripheral blood were analyzed with anticoagulant (2 mL), coming from dogs that showed nervous signs compatible with DC and that in some cases presented positive to the ELISA test specific for CDV, with IgM antibody titers ≥1: 80. These samples were collected from veterinary clinics of Santiago de Chile, maintained in refrigeration at 4 °C and processed in less than two weeks. In addition, they were classified according to race, sex, age, vaccination status against CDV and anti-CDV IgM antibody titers (Table 1 )

Table 1: Classification of peripheral blood samples from dogs with suspected nervous signs of CD, according to: race, sex, age (y=year; m=month), vaccination status against CDV and anti-CDV IgM antibody titers.

Obtaining the phlogistic layer

In blood samples with anticoagulant, negative controls and animals suspected of DC, the preparation “Histopaque®-1077” from Sigma-Aldrich® was used according to the manufacturer’s instructions, for the isolation of lymphocytes and other mononuclear cells, which are the target of the CDV infection, so a higher concentration of viral RNA was obtained in the samples where the virus was found. For this, 2 mL of Histopaque®-1077 were placed in centrifuge tubes, adding 2 mL of blood with anticoagulant, and then centrifuging at 400xg for 30 minutes at room temperature (RT). The mononuclear band (opaque interface) was then carefully transferred with a Pasteur pipette to a clean centrifuge tube, washed with 10 mL of saline, shaken and centrifuged at 250xg for 10 minutes, to then aspirate. The supernatant and discard it.

Viral RNA extraction using “Trizol LS” kit from Invitrogen®

750 μL of Trizol reagent was added to the mononuclear blood cells, obtained as described above and to 250 μL of the positive vaccine controls (blood contaminated with vaccine), incubating for five minutes at RT. Then, 200 μL of chloroform was added to each tube, they were mixed vigorously for fifteen seconds and incubated at room temperature for five minutes. Then, they were centrifuged at 7000xg for fifteen minutes and the aqueous phase was transferred to a clean tube. For the precipitation of the RNA, 0.5 mL of isopropanol was added, left at room temperature for ten minutes, centrifuged at 7000xg for ten minutes, the supernatant was removed, washed three times with 75% ethanol (1 mL), fifteen were shaken seconds in vortex and centrifuged at 2000xg for five minutes. For the resuspension of the RNA the supernatant was removed, the RNA precipitate was dried under vacuum for five minutes and resuspended in 100 μL of nuclease-free water. Finally, the RNA was incubated at 55-60° C for ten minutes and kept at -20 ° C for later use.

RT-PCR

A 96-well, 96-well Apollo thermocycler (CLP, USA) and a protocol with appropriate temperatures, times and cycles for each stage were used. The primers for the RTPCR were sent to be synthesized to the Bioscan® company, their sequences being in a highly conserved region of the nucleocapsid gene: P1: 5’-ACAGGATTGCTGAGGACCTAT-3’ and P2: 5’-CAAGATAACCATGTACGGTGC-3’. These primers allowed to amplify a fragment of 290 base pairs (bp).

RT-PCR reaction

This was done using the “SuperScript ™ one step RTPCR with platinum Taq” kit (Invitrogen®) according to the manufacturer’s instructions. The reaction began with the synthesis of complementary DNA and a pre-denaturation performed in one cycle (45°C for thirty minutes and then two minutes at 94°C). The PCR amplification was carried out in forty cycles, which consisted of a denaturation (94°C for one minute), alignment of the starters (60°C for two minutes), elongation (72°C for two minutes), and a final extension in one cycle (72°C for ten minutes).

Detection of the DNA fragment synthesized in the RTPCR

The RT-PCR products were analyzed by means of 2% agarose gel electrophoresis in Tris-HC1 buffer (100 mM Tris-HC1, 10 mM EDTA) and comparison against a molecular size standard: Hyperladder IV (Bioline®). The PCR product was mixed with a commercial loading product (Fermentas®) and then the electrophoresis was carried out at 90 V for forty-five minutes. After electrophoresis the gel was incubated with ethidium bromide (0.5 μg/mL) for thirty minutes, then visualized in an ultraviolet light transilluminator and subsequently photographed.

Sequencing of Amplified Fragments

The fragments of DNA obtained from two positive samples were sent to be sequenced, to the Sequencing Center of Genytec Ltda

Bioinformatic analysis of the nucleotide sequences of DNA fragments

The sequences obtained were aligned using the free online program Clustal Ω for obtaining a consensus sequence for each sample used. In addition, the consensus sequences were entered the BLAST program to identify the origin of the DNA fragments obtained in the RT-PCR.

Analysis of results

Those samples that after RT-PCR synthesized a DNA fragment of approximately 290 bp were considered positive and after analysis with the BLAST program their nucleotide identity corresponded to CDV.

ResultsImplementation of the RT-PCR assay of the CDV Nucleocapsid Protein Gene

Once the RT-PCR protocol of the nucleocapsid protein gene of the CDV was established, the operation of the method was tested with positive and negative controls, to later analyze the field samples suspected of DC

Controls

Figure 1: Visualization of products amplified by RT-PCR by electrophoresis in 2% agarose gel and subsequent incubation in ethidium bromide. Lanes 1 and 2 correspond to the negative controls, unvaccinated dog vaccinated and without vaccination respectively. Lanes 3, 4 and 5 correspond to positive vaccinal controls, negative control blood without vaccination contaminated with Onderstepoort, Lederle and Snyder Hill vaccine strains, respectively. Lanes 6 and 16 correspond to the molecular size marker (100-1000 bp). Lanes 7, 8, 9, 11, 12, 13, 14, 15, 17 and 18 correspond to the positive controls of viral RNA from national isolates to CDV.

Three positive controls from commercial vaccines, ten positive controls corresponding to CDV RNA (national isolates) and two negative controls from uninfected dogs were processed. All these controls were subjected to the RT-PCR of the nucleocapsid gene of the CDV and after the visualization of the products, all the positive controls generated intense bands of around 290 bp and in the case of the negative controls, they did not present visible bands (Figure 1 ).

Suspicious samples of DC

Eleven samples of peripheral blood of dogs with nervous signs concordant with DC were collected and, in some cases, they were positive to the specific ELISA test for CDV (IgM) (Table 1 ). All these samples were submitted to the RT-PCR of the nucleocapsid gene of the CDV and after visualization of the products, ten samples (canine 1 to canine 10, Table 1 ) presented bands of around 290 bp, the expected DNA fragment. On the contrary, the sample from canine 11 did not generate visible bands (Figure 2 ).

Controls

Figure 2: Visualization of amplicons by RT-PCR by electrophoresis in 2% agarose gel and subsequent incubation in ethidium bromide. Lane 1 are the positive vaccine control, Onderstepoort strain. Lane 2 is the negative control, uninfected dog without vaccination. Lane 9 and 16 are the molecular size marker (100-1000 bp). Lanes 3, 4, 5, 6, 7, 8, 11, 12, 13 and 14 are blood samples from canines 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 respectively. Lane 15 are canine 11 (Table 1 ).

Sequencing of amplified fragments and bioinformatic analysis of the nucleotide sequences of the DNA fragments

The DNA fragments from two samples considered positive (canine 1 and canine 3, Table 1 ), were sent to be sequenced in triplicate to the Sequencing Center of Genytec Ltd., obtaining five sequences for each sample. The selection criteria for these samples was based on the ages of the animals, where the six-year-old canine 1 was chosen, which is the age where chronic encephalitis would most frequently occur due to CDV infection, which could be related to greater nucleotide variations according to [7]. Likewise, dog 3, eleven months old, was chosen, which is the age in which more animals are usually infected by CDV (less than one year old). The sequences were then aligned using the Clustal Ω program, obtaining a consensus sequence for each sample used: from canine 1 the sequence CDV/CMC1 was obtained and from canine 3 the sequence CDV/CMC2 (Annex 1) was obtained. Next, the consensus sequences were entered into the BLAST program, to identify the origin of the DNA fragments obtained in the RT-PCR, which revealed that the sequence CDV/CMC1 presented a percentage of nucleotide identity of 95% and the CDV/CMC2 sequence of 94%, with respect to the first one hundred results corresponding to CDV, and that in the case of both sequences, these corresponded to CDV (Annex 2).

Discussion

The CD is a disease of worldwide distribution, viral and highly contagious, affecting a wide range of terrestrial carnivores and some marine mammals, which have had a progressive increase in recent years, infecting even animals with their vaccination plans at day. It is due to this increase in hosts, that a true diagnosis of the disease caused by the CDV is crucial, to limit the spread of it to other animals and to give an opportune treatment to the sick. Although the diagnosis of CD is based mainly on the clinical signs of the patient, in certain occasions, it is difficult to reach a final diagnosis in this way, since the main respiratory and digestive signs are common to other diseases that affect the canines and in the case of the more specific clinical signs of DC, such as nervous signs, these most commonly occur in the later stages of the infection, after it has become more widespread. Also, the different clinical presentations of DC vary from one animal to another, making the final diagnosis more complex [1,10]. Although there are several techniques to help the diagnosis of CD, such as virus isolation, immunofluorescence and ELISA, most of these methods are laborious, time-consuming and may give false results, which is why they are convenient for the definitive ante-mortem diagnosis of the disease. For this reason, a sensitive, specific and rapid method is necessary to detect a small amount of the virus early in the infection, which, thanks to the development of molecular biological techniques such as RTPCR, may be possible, where a good white sequence is required. conserved between different strains of the CDV, to avoid mismatches of the splitters and the subsequent failure to amplify it.

In this work a diagnostic method for CDV was implemented through RT-PCR, based on the detection of the nucleocapsid protein gene, a highly conserved region of the CDV genome, which makes this an excellent target gene for the molecular detection of this virus. In fact, several authors have revealed the suitability of molecular biology methods based on the nucleocapsid protein gene for the detection of CDV [1,6-8,12,15]. Additionally, the primers used in this study are in the most conserved region of the nucleocapsid protein gene, the central zone, which would increase the sensitivity of the method [7,8].

Although two Faculty reports based on the CDV RT-PCR have been carried out in our Faculty [13,14], these were based on the hemagglutinin gene, which has the highest antigenic and genetic variation among the CDV genes, presenting about 10% variability between different strains of CDV, which is not the most suitable gene for the diagnosis of the virus [8,11].

In the present study, the previous observations of the RT-PCR utility of the CDV nucleocapsid protein gene were confirmed, with the virus RNA found in about 91% (10/11) of the field samples suspected of DC (Figure 2 ). This result suggests that the protocol used in this title memory, from the obtaining of the phlogistic layer to the visualization of the amplified products, could be highly sensitive. At the same time, this method would be more sensitive than the protocols implemented in previous studies, since [13] only found viral RNA in 7% (3/42) of the samples analyzed that came from dogs suspected of DC and in the case of [13], he found the RNA of the virus in 83% (5/6) of the samples analyzed, which had previously been confirmed as positive by another RT-PCR, however in twenty field samples of dogs suspected of DC, did not find viral RNA [13]. However, to corroborate the sensitivity of the method implemented, additional studies are required. The specificity of the method was corroborated with the amplification of the expected fragment in 100% (10/10) of the positive controls and in the nonamplification of the fragment expected in the negative controls (Figure 1 ). As for the negative controls, it is important to note that one of the animals used for these controls, had its vaccination schedule up to date and had been vaccinated for the last time ten months ago, which supports the preliminary observations that a previous vaccination does not causes false-positive results [7].

On the other hand, CDV can remain active indefinitely at -70°C, -192°C (liquid nitrogen) or lyophilized and only remains active for about a month at -10°C [16], without However, in the positive controls corresponding to CDV RNA (coming from national isolates), it is important to note that this RNA had been stored for more than a year at -20 °C, which is not the ideal temperature to maintain integrity and not degradation, but despite this, the stability of the virus RNA was not affected. Regarding the type of sample chosen for the field samples, blood was chosen with anticoagulant (ethylenediaminetetraacetic acid “EDTA”), because it is one of the most suitable as a substrate for RT-PCR, since it is an easy sample of obtain, maintain cell morphology for a longer time preventing the viral RNA from being degraded by endogenous RNAses when leaving the cell and also the amount of EDTA present in the tubes to obtain blood, would not inhibit the result of the RT-PCR [17]. However, similar results have also been seen with serum and urine samples, further increasing the sensitivity of detection if more than one is used for the diagnosis of the disease [1,7].

Until now, confirmation of CDV infection in live dogs was not useful, mainly due to the low level of sensitivity of the available methods, such as the ELISA test, where the finding of neutralizing antibodies in general does not correlate with the results of the RT-PCR [7], as in this study, where some animals positive for CDV infection had lower antibody titers than those considered positive (<1:80); which could indicate the non-contributory role of neutralizing antibody titers for the etiological diagnosis of the disease.

The field sample suspected of DC that was negative after being analyzed by the proposed method (canine 11, Table 1 ), could have been due to the absence of viral RNA, since either because the virus was not in blood, it could be in other epithelia of the body or because the sample was poorly preserved or poorly processed.

Although DC disease is less frequent in developed countries due to immunization, in recent years, the virus that produces it has emerged as a significant pathogen of vaccinated animal populations, and in the case of this study, the presence of some animals suffering from DC, which were at least once vaccinated against the virus, which raises the hypothesis of failure in the immunization of the vaccine, which could be due to incorrect vaccination plans or genetic variations of the virus [6], however, in order to respond to these presumptions it is necessary to carry out other studies on the subject.

Annexx 3:

After obtaining the sequences of two of the samples that amplified a fragment of approximately 290 bp and defining the consensus sequence for each of them (Annex 1), the BLAST program was used, where it was obtained that both sequences presented a percentage of nucleotide identity greater than 94% with respect to the first one hundred results corresponding to CDV and that in both cases the amplified fragments belonged to this virus, corroborating the specificity of the method implemented (Annex 2).

Annexx 4:

The technique implemented, would allow the early isolation of infected animals and the establishment of an appropriate treatment. In addition, in the case of wild animals, the rapid and specific diagnosis given by this method could help the conservation of protected species, which have been at risk due to the dramatic increase in CDV hosts occurred in recent years.

Conclusion

The implemented system represents a fast and specific antemortem diagnosis method of the Canine Distemper disease, being effective for the detection of the virus. The stability of the RNA of the Canine Distemper Virus was not altered when kept for more than one year at -20 °C.

According to the genomic analysis of the sequences CDV/ CMC1 and CDV/CMC2, both showed greater similarity to the Onderstepoort strain of the Canine Distemper Virus and, on the other hand, both sequences showed greater similarity to the American 1 lineage of the Canine Distemper Virus

Based on the results of this study, the implemented method can collaborate with the prevention and control of the increase in Canine Distemper, since it provides a rapid and specific diagnosis for the identification of the responsible virus, which could help improve health of the dog population, as well as that of other animals that are susceptible to the disease.

Read More About this Article: https://biomedgrid.com/fulltext/volume5/the-nucleocapsid-protein-gene-as-excellent-target-for-detection-of-canine-distemper-virus-by-reverse-transcriptase-polymerase-chain-reaction.000935.php

For more about: Journals on Biomedical Science :Biomed Grid | Current Issue

[districofcolumbiaprelawland-blog]

The Evolution Of The Fourth Amendment: From Protecting The Public To Protecting The Police

By Jacob Prince, American University Class of 2021

February 23, 2021

The Fourth Amendment details the protections that civilians have in relation to government entities. It protects them from “unreasonable searches and seizures” without a proper and valid warrant. [1] The protection from search and seizure derives from English law, prohibiting the monarchy from infringing on their subjects’ private property. The same concept was applied to the United States in the 18th century, which limited the federal government’s authority over one’s land or possession. In the present day, protection from unlawful search and seizure is most commonly applied to the power that the police have over the general public. However, the Fourth Amendment does not protect people from use of force, either reasonable or unreasonable. Meanwhile, the application of the Fourth Amendment has been weakened since its implementation in the Bill of Rights. Supreme Court rulings have given more authority to the police force to exert searches and seizures without a proper warrant. Should the court system continue to use the Fourth Amendment as the legal backbone of police use of force and misconduct?    

In the case of Terry v. Ohio in 1968, the Supreme Court set the precedent of stop and frisk, the concept that police can search anyone deemed suspicious, as not unconstitutional. The practice of stop and frisk allows the police to search an individual without a warrant or probable cause for an arrest. [2] Terry v. Ohio preceded several other Supreme Court rulings in favor of expanding on police powers. In the 1983 case Michigan v. Long, the Supreme Court extended stop and frisk to traffic stops with any sense of suspicion. [3] Outside of stop and frisk policy, the courts have authorized the power to search without warrants in special cases. For instance, the case United States v. Robinson in 1973 concluded that a viable arrest nullifies the need for a warrant to search the individual under arrest. [4] 

The Fourth Amendment has evolved to include the protection against unnecessary and excessive use of force by the police. However, the courts empower the police to use some degree of force and to threaten the use of force. These two principles were decided by Graham v. Connor of 1989, which created the reasonableness test for evaluating the reasons behind the actions of a police officer when using physical force. [5] Despite the fact that these court decisions grant the general public rights when interacting with the police, it also paved the way for police officers to be acquitted more commonly in cases involving police misconduct. The Supreme Court adopted the theory “totality of circumstances” to determine excessive force by police officers, but the theory is rudimentary in real practice. [6] Rhetoric constructed to outline necessary and unnecessary use of force by the police is convoluted, and only aids the police in cases of excessive physical force. [7] An example of this is the case of Clabrook v. Birchwell, brought to the Court of Appeals for the Sixth Circuit. Quintana Claybrook, a third party to the event, was shot during police intervention, but the Fourth Amendment was inapplicable to the case, according to the court. [8] It is evident that the Fourth Amendment is cited for misconduct and illegitimate use of force by the police, but rarely does it protect people from use of force or physical seizure.  

In recent decades, government interference in personal technological devices and private data has entered the discussion pertaining to the Fourth Amendment applicability. President George W. Bush’s Patriot Act of 2001 served as the catalyst of widespread government surveillance into the general public’s private phones after the terrorist attack of 9/11. The law expanded law enforcement’s ability to tap into phones, including phone calls, text messages, and other forms of electronic data. [9] Critics of the Patriot Act argue that the law is unconstitutional, directly conflicting with the Fourth Amendment’s improper search and seizure principle. Government interference in private data is controversial since personal phones now contain sensitive information, including private conversations, financial information, private photos and videos, and other sensitive material.  However, the Supreme Court ruled in favor of the Fourth Amendment in the Riley v. California case in 2014, which prohibited police from unlawful search and seizure of personal phones.[10] In addition, the court ruling of Carpenter v. United States in 2018 strengthened the Fourth Amendment to include phone locations, called cell site location information. [11] 

Despite positive efforts to protect personal devices, courts have ruled in support of law enforcement around the southern border of the United States. Border Patrol agents have been given increased authority to search people’s belongings and phones without a warrant. The United States Court of Appeals for the First Circuit concluded that searches conducted by Border Patrol agents without a proper warrant are not unconstitutional, citing the need to protect the interests of the country’s security. [12] In the decision, the court ruled that the jurisdiction of Border Patrol agents should extend from 100 miles away from the north and south borders and along the coasts. 200 million people live within 100 miles of borders and coasts, with cities like New York City and Los Angeles included in these zones. Border agents can conduct routine searches of anyone they deem necessary in these areas, with cell phones and other belongings susceptible to searches. [13] 

Though the Fourth Amendment was drafted to give protections to the citizens from unlawful searches and seizures, recent history has convoluted these protections. Court interpretations of the Fourth Amendment have overwhelmingly awarded law enforcement entities more authority to search the general public’s property and to use force. The court rulings have also created more room for immunity for police officers and other law enforcement personnel. The landmark Supreme Court rulings granted law enforcement more protection to continue their use of stop and frisk tactics, physical use of force, and to search personal technological devices.  

______________________________________________________________

[1] The Constitution of the United States, Amendment 4.

[2] Terry v. Ohio, 392 U. S. 1 (1968)

[3] Michigan v. Long, 463 U. S. 1032 (1983)

[4] United States v. Robinson, 414 U.S. 218 (1973)

[5] Graham v. Connor, 490 U. S. 386 (1989)

[6] Illinois v. Gates, 462 U. S. 213 (1983)

[7] Seth W. Stoughton, How the Fourth Amendment Frustrates the Regulation of Police Violence, 70 Emory L. J. 521 (2021).

[8] Clabrook v. Birchwell, 2000 FED App. 0014P (6th Cir.) 

[9] Public Law 107–56: Uniting and Strenghenig America by Providing Appropriate Tools Required to Intercept and Obstruct Terrorism (USA Patriot Act) Act of 2001. (115 STAT. 272; Date: 10/26/01; enacted H.R. 3162)

[10] Riley v. California, 573 U. S. 373 (2014)

[11] Carpenter v. United States, 585 U.S. 138 (2018)

[12] Alasaad v. Mayorkas, No. 1:17-cv-11730-DJC (D. Mass Aug. 14, 2020)

[13] ACLU, The Constitution in the 100-Mile Border Zone, https://www.aclu.org/other/constitution-100-mile-border-zone.

[antarestyl]

That last one sounds very final, almost like the end of the candy saga... So maybe after the flames and skeles go back to the underground, Kitty is old enough and the candy kingdom has her as queen Cuz they all love her... And two letter end up in the underground, delivered to the flames (mainly Fellby) And one is like Kitty thanking him and the flames for raising her- Kitty C Sugarplum And the other is an apology, like “I’m not going to explain, it’d be an excuse.” And a Thanks- CVS

Yep sounds like a good end to that chapter. XD

Of course it wouldn’t be the end of the Candy Saga. It’s just a new start. ^^ (Maybe they will be redemption for Chesire even. It’s a long time this takes place so there are a lot of things possible) but I like that “I am not going to explain, it’d be an excuse” letter. *nods* I can see this.

But yeah, I still think the flames and kids will visit candy land from time to time still, even when they are living on the surface after Frisk went through the Underground. Just like… visits. (the boys have to visit their little sister after all. ^^)

And well, in the TBS AU, flames and other elementals live like a VERY long life. So the flames have all the time in the world to watch the Candy land prosper. ^^

And well, like I said, there are a lot of years between the start of the Candy Saga and the end of that first chapter of it.

(Let’s see… Sans is the oldest with around 9 years old at the start of the TBS AU timeline… It might be a few years until we start the Candy land adventures… let‘s say it’s after some important plot points so Sans would be around 15 years old. And at that point Kitty is a toddler so let’s say 3 or so. So it would take at LEAST 15 years from then until the flames leave the crown to Kitty… so 15 years worth of the Candy King Fellby Saga. ^^)

I dunno if Kitty would call herself Queen thou. It could be the Candy people don’t connect genders to titles (like Swabby and Grillby both got the title Queen) so Kitty would in fact be King (or she would give the King title to somebody else and be a Queen because like that she would have a bit more power? Who knows? ^^ Or Fellby says King but they change it so that Kitty kinda can do everything when Fellby is not around? So many possibilities. ^^)

[cochrancaliforn]

Racial Profiling Law Services: A Relief To The Victim Of Discrimination

Racial Profiling Law service is the exercise of targeting particular people because of their ethnic background or race. It is the practice followed by law enforcement officers of using race and national origin as a salient basis for suspicion of criminal activity. Police officers are racially profiling when they view people as suspicious because of who they are, what people look like, rather than what they have done. 

 Basis

Racial Profiling performed based on the following factors:

Race

Nation Origin

Ethnicity

Religion

  Examples

Law officers are pulling over a minority driver because     they are driving an expensive car.

When the police see a group of African American males     walking down the street, and they stop and frisk them under the mistaken     belief that they are selling drugs.

If a police officer stops a person, they believe to be     of Middle Order descent under the assumption that they are planning a     terrorist attack.

 Quantitative Evidence

v Black Americans and White Americans are drug users at a similar rate, but Black Americans are six times more likely to be arrested for it. 

v One US study founds that job cv's with traditionally white-sounding names received 50% more callbacks than those with historically black names. 

v Black Women are 3 to 4 periods more likely to experience a pregnancy-related death than White Women, even at a similar level of education and income. 

v In the US, Black workers are less likely than White workers to be employed in a consistent job with their level of education.

 Effects

Depression

Mental disorder

Physical and mental stress

Human Rights exploitation

Physical injuries

Fear in community

 General tips of prevention

Focus on diversity

Educate the public

Write or review your policy

Communicate

Build your team 

 Legal facts 

Racial Profiling is against the laws of California and the US. The law states that the person targeted by a police officer for their skin color, race, national origin, and so on are victims of discriminatory practices. US constitution provides equal protection to every American and also prohibits unreasonable searches and seizures. 

Lawyer services

Racial Profiling attorneys are working tirelessly to ensure the satisfaction and peace of the mind of the victims. They represent their clients in courts and use all their resources to claim or to fetch the justice and compensation amount they deserve. The compensation amount includes medical expenses, income loss, future income loss, pain, and sufferings. We ensure the safety of the victims and their loved ones from the hectic and stressful legal issues. 

 Summary 

In recent years, Racial Profiling becomes a more complex and hectic legal issue for various persons. Most probably, the police officers are misusing their forces for troubling the civilians based on their race. It is an illegal exercise, and any person who is facing this comes under discriminatory factors. The victim has the right to file a case against the defaulted party and fetch a positive outcome for the pain and suffering he faced.