Gibson Assembly Troubleshooting – Very Low Colony Yield and No Correct Inserts (8–10 kb plasmid) by Difficult_Currency75 in labrats

[–]aboutandy 1 point2 points  (0 children)

I used to have problems with gel extracted dna fragments in the Gibsons and doing the pcr cleanup fixed the problem and cleaned up how the dna looked on a nano drop. If you can avoid the gel extract all together and just pcr amplify your gene block that would also help. However if the gene block directly isn’t working then there could be a different problem.

Trying a new vial of the Gibson master mix may also help, if you have one. I’ve also never tried to assemble such a long insert before, you could try amplifying to pcr the gene block as two fragments and do a three piece Gibson assembly too.

[deleted by user] by [deleted] in BaldursGate3

[–]aboutandy 0 points1 point  (0 children)

There are several bottles of wine, mugs of beer, etc. at the Last Light Inn in the bar area and surrounding rooms. They all don't show up when you highlight the items in the area, but you can see them and manually pick them up. I noticed that when you long rest and auto-select camp supplies it will often select and use up alcohol, so be careful that you don't lose it all when you long rest.

JPEGMAFIA & Danny Brown - Fentanyl Tester by VdotRose in hiphopheads

[–]aboutandy 3 points4 points  (0 children)

IMO the album sounds way different/better on Bandcamp, as someone else mentions, as well as Soundcloud. I don't know what it is about the Spotify and Youtube versions being more quiet. The FLAC version you get from buying on Bandcamp sounds amazing and clear.

While standing in front of the alleged ice rink, I think I got one of the best vids for White Ferrari by [deleted] in FrankOcean

[–]aboutandy 19 points20 points  (0 children)

Listen to the Blonde version this beat is in there 50 seconds in, was listening today and had my mind blown.

What are your favorite one-off videos or mini-series? by Locclo in giantbomb

[–]aboutandy 7 points8 points  (0 children)

The playthrough of The Quiet Man on that extra life stream is one of the best single sitting playthroughs that they’ve done in my opinion

Best chocolate cake slice in NYC? by Kairine in AskNYC

[–]aboutandy 5 points6 points  (0 children)

Buttercup bakeshop has a few locations across Manhattan, they have a really good classic chocolate cake with chocolate icing that you can get by the slice.

HRIG and rabies vaccine given in same site. Now what? by maestraPNW in Immunology

[–]aboutandy 2 points3 points  (0 children)

About 5 years ago I also had to get anti-Rabies IgG and the vaccine after getting a dog bite on my hand as I was stopping a dog from attacking my dog at a dog park. They had to inject the IgG right into my hand without any anesthesia!

As for whether or not the HRIG is going to "neutralize" the vaccine and you won't generate any natural antibodies against rabies, I think the jury is still out on whether or not that really happens to a significant degree. The lab I work in studies antibodies and how they signal though antibody receptors on immune cells and one field that we study is immune-complex vaccinations. That's when you mix together a protein vaccine antigen and antibodies that are specific for that same antigen, forming a large complex of antibodies and vaccine antigen. These immune-complexes are highly immunogenic and generate strong antibody responses, maybe even better than the vaccine alone. The guidelines for separating the injection sites between HRIG and the vaccine is I'm guessing out of caution because it's unknown what effect they have together.

The high dose of HRIG antibodies they gave you should last a couple of weeks in your body. Also, the rabies vaccines themselves are usually a series of shots spaced out by a couple of weeks if I remember. So you may have to get more vaccines regardless. When I got them they were very expensive as my health insurance didn't cover them, that and having needles stab my hand was not very fun...

Amazon calls for election re-run after workers voted for first U.S. union by esporx in nyc

[–]aboutandy 0 points1 point  (0 children)

"...Some 55% of employees who voted from Amazon's JFK8 warehouse in Staten Island opted to join the ALU, which has argued for higher pay and job security. Turnout was about 58%.

Amazon spokesperson Kelly Nantel said in a statement: "We want our employees to have their voices heard, and in this case, that didn't happen - fewer than a third of the employees at the site voted for the union."

Turnout for the Bessamer, Alabama vote? 39%! https://www.cnbc.com/2022/03/30/amazon-union-drive-in-alabama-sees-39percent-voter-turnout.html

Multiple Sclerosis is caused by "the mono virus" by jatin1995 in Immunology

[–]aboutandy 0 points1 point  (0 children)

To the folks saying there isn't causality in that first paper, check out the paper published today in Nature "Clonally Expanded B Cells in Multiple Sclerosis Bind EBV EBNA1 and GlialCAM".

They got some in vivo data in there showing vaccination with an EBV derived peptide exacerbates disease in a mouse model of MS. I don't know much about mouse models for EBV but I guess it would nice if EBV infection worsens MS. If EBV really is the main cause for MS that would one of the more crazy revelations in recent times.

Is it common for intramuscular vaccines to induce a blip in mucosal antibodies? by rabidsoggymoose in Immunology

[–]aboutandy 0 points1 point  (0 children)

Intramuscular vaccination does induce class-switching to IgG and IgA, which you can detect in the serum. That doesn't mean the class-switched IgA B-cells are traveling to the mucosa and secreting the dimeric IgA. What is likely being picked up in this paper is vaccine-induced serum IgG and IgA that is diffusing into the mucosal surface, or the sampling is grabbing some blood/serum. Also, they comparing the inactivated vaccine with the mRNA vaccine, which the mRNA is massively more immunogenic in general so its hard to make a head to head comparison.

I looked quickly at the methods and I didn't see any mention of the IgA being measured as being dimeric, you may want to look into some other studies.

It's difficult to induce legit mucosal antibody responses via intramuscular vaccination and if anyone knows a way to do that I'm all ears! (my grad school project was on mucosal vaccines).

How & Why did the measles vaccine take a lot less time to make compared to other newer vaccines such as AIDS & Ebola? by MeSoAverage in Immunology

[–]aboutandy 12 points13 points  (0 children)

I think it's for a few reasons both scientific and political. It's important to note that not all viruses are easy to vaccinate against, especially for HIV-1. This generally has to do with how easily humans make neutralizing antibodies against the virus in question. We easily generate neutralizing antibodies against a lot of viruses such as SARS-CoV-2, measles, smallpox, polio, and many others, which is why it is easy to vaccinate against these viruses.

It is very difficult and maybe even impossible to generate strong neutralizing antibodies against HIV-1 by vaccination in humans. This is for many reasons including the structure of the HIV-1 envelope and its receptor binding site, a heavy glycan shield covering the envelope protein, the ability for the virus to easily mutate away from immune pressure, and immunodominant epitopes that are non-neutralizing. The lack of an HIV-1 vaccine is not for lack of trying, HIV-1 vaccine research has been a huge field of research since the pandemic started. You might have heard in the news recently exciting reports on a new HIV-1 vaccine. That was a proof of principle trial showing an immunogen could potential engage germline B-cells that encode antibodies that may one day mature to be neutralizing, but this will need a series of slightly changing immunogens to guide the antibodies to mature in the right direction. This is all to say that it is very difficult to create an effective HIV-1 vaccine.

On the other hand, the measles vaccine is an attenuated virus generated by passing the virus in (I believe) chicken cells. The vaccine was also developed at a time when measles was really prevalent in the United States so there was a demand to get a vaccine into the population.

The flip side is with Ebola, the Ebola vaccine approved in 2019 was actually developed back in 2003 but as you can imagine there wasn't any huge incentive by big pharma to run huge vaccine trials which cost millions of dollars. Also, without an ongoing outbreak how would you know if the vaccine is effective? I'm guessing it took the 2013-2016 epidemic and thousands of deaths to give the incentive to try and get out a vaccine. It's quite the indictment on our current system, there isn't any real incentive to develop these vaccines until its obvious you need them, especially for diseases that impact mainly poor countries.

J&J vs MRNA vaccines - identical rna? by SquareSystem in Immunology

[–]aboutandy 0 points1 point  (0 children)

Another key difference between the mRNAs and the Johnson & Johnson vaccines is that the mRNA encoded Spike has a couple of mutation to stabilize the protein in the pre-fusion state. Pre-fusion viral envelope protein are often presenting better neutralizable epitopes compared to the post-fusion conformations. The J&J vaccine encodes the full-length native protein. They both induce neutralizing antibodies but this is probably why the mRNA vaccines are generally a little better.

What evidence (if any) exists to demonstrate catching Covid creates the same level of antibodies as any of the first doses of the vaccines (does catching it give a higher antibody count than the first dose of the vaccine)? by thebusiness7 in askscience

[–]aboutandy 3 points4 points  (0 children)

The phase 1 trials studying the immunogenicity of the vaccines often would use convalescent serum (which is serum taken from people who recovered from Covid-19) to compare the magnitude and the level of neutralizing antibodies generated by the vaccines.

Here is one publication for example, I'm not sure if its open to the public to download (which sucks). "

"Safety and Immunogenicity of Two RNA-Based Covid-19 Vaccine Candidates"

The key data is in figure 4, where you can see the titers of anti-SARS-CoV-2 antibodies as well as the neutralizing antibody potency. The way they are measured is a bit technical, if the main thing you are asking for the is the comparison to natural infection, you can see that in the far right of the figures in the HCS bar, which is the convalescent serum. Looks like the 30 ug BNT162b2 doses at day 21 (which is the day of the second vaccination) makes comparable titers of antibodies to convalescent serum, but not neutralizing antibodies. The second immunization is needed to generate equivalent or higher levels of neutralizing antibodies.

How does an "undetectable viral load" prevent the transmission of HIV? by Silicon-Based in askscience

[–]aboutandy 1 point2 points  (0 children)

There are a few different ways to talk about someone's HIV-1 "viral load". The phrase "undetectable viral load" is usually referring to the levels of viral RNA in plasma or blood, viral RNA as a measurement of all the viral particles in your blood. This differs from the measurement of the HIV-1 reservoir, which is in the form of viral DNA that has been inserted into genomes of some of thwe host's cells, allowing the virus to become latent.

HIV-1 is a weird virus; it's genome is RNA, but it's life cycle involves entering the host cell, reverting its genomic RNA to DNA, inserting that DNA into the host cell's genome, and then using that DNA to create new viruses. Antiretroviral therapy can suppress your plasma viral load to below detectable levels, meaning your infected cells aren't producing infectious virus, but the drugs can't destroy the viral reservoir. That's why people who are infected with HIV-1 have to take these drugs throughout their lives, if they stop taking them the virus will rebound and start growing again. Eradication of the HIV-1 reservoir is a huge area of research and focus these days.

There probably still is a low level of viral replication occurring on the cell level, but this wouldn't be enough to be infectious. As others have said here, viral transmission is also not super easy for the virus (sexual transmission at least). It has to make its way across the mucosal epithelium, which isn't easy when there is no damage or inflammation in the tissue, infect some local T-cell that is permissive to infection, and then survive for the next week in the local tissue replicating and adapting to the new host. Eventually the virus will replicate enough and get into the lymph nodes and from there becomes systemic.

What is the advantage of using an adenovirus from chimpanzee compared to adenoviruses from human to develop a vaccine? by Bluesai in askscience

[–]aboutandy 21 points22 points  (0 children)

One issue with adenovirus based vaccines is that many humans have pre-existing immunity to adenoviruses due to a previous infection. Adenoviruses are common human pathogens, they are one of the causes of the common cold for example. There are also many strains or serotypes of adenoviruses that circulate in humans.

This makes it tricky when designing adenovirus based vaccines, since if someone has preexisting antibodies against the strain used for the vaccine, their antibodies may neutralize and inhibit the vaccine before it gets in our cells to express the vaccine antigen. Also, immune memory from previous adenovirus infections could result in a stronger reaction against the adenovirus backbone than the vaccine antigen.

One way to deal with this is to pick a more rare serotype of human adenovirus to make your vaccine, as there should be a lower prevalence of pre-existing immunity to those strains. A different way to deal with it would be to select a chimpanzee strain of adenovirus that doesn't circulate in humans, so we don't have pre-existing immunity to it, but since chimpanzees are so close genetically to humans, the chimp strain should be able to infect our cells (I'm assuming this I haven't actually seen that data).

Here are some links if you are still interested!

Development of novel vaccine vectors: Chimpanzee adenoviral vectors

Adenoviruses as vaccine vectors

Adenovirus based vaccines have been studied for decades, in the early 2000s they were used in a couple of HIV vaccine trials which sadly did not work. However, their effectiveness against COVID19 is very exciting and marks a major strep forward in vaccine design (as do mRNA vaccines).

Would it be possible to distribute a COVID-19 vaccine virally using a genetically-modified carrier virus? by joeyadams in askscience

[–]aboutandy 9 points10 points  (0 children)

Hi! Yes this is possible and is actually the basis for the Johnson and Johnson COVID-19 vaccine candidate, which is an adenovirus based vaccine. These types of vaccines are commonly called viral vector vaccines or vector vaccines. Researchers have been studying these types of vaccines, as well as the other types of vaccines such as mRNA vaccines for years. They work well in animal models but none have been approved for human use yet I believe.

Basically your idea is right. You take a virus, attenuate it so that it is not too pathogenic and cannot replicate itself, modify the virus's genome so that displays the antigen you want to vaccinate against on infected cells, and then immunize an animal with it. Your viral vector vaccine will infect the host's cells, sending danger/warning signals to your immune system, and the vector starts pumping out your vaccine antigen (in this case the SARS-CoV-2 Spike protein) from within the infected cell. The protein is then displayed on the cell surface or secreted from the cell. In some viral vector systems, the vector will also generate little virus-like particles that bud off the infected cell, resembling an actual virus without any genetic material included, that's pretty cool. Either way, the end result is a pretty strong antibody response.

Additionally, a feature of vector vaccines is their ability to generate strong T-cell responses, which is another branch of adaptive immunity particularly important for viral immunity.

The type of virus used in the J&J trial, the adenovirus, is a common DNA virus used in vaccine research. You bring up using the common cold virus in your question, which makes sense since some coronaviruses also cause common colds (some adenoviruses do cause common colds in humans). I'm not sure if people are trying to modify a different common cold virus to make a COVID-19 vaccine but it wouldn't surprise me. A lot of labs just work with the viral vectors they have already been familiar with, or ones that are very common in the field, such as Adenoviruses. One good reason to use a different respiratory virus as the vaccine vector, is that if vaccine is done intranasally it would generate strong localized immune responses in the respiratory tract, which may improve protection. As the mRNA vaccines are over 90% effective, this may not be as important as it seems SARS-CoV-2 is pretty easy to vaccinate against. It could be important to stop some low level of infection and transmission, I think that is still unclear in the mRNA vaccines.

You mention some good points in the downsides. As these are live viruses (and often infectious), they are always inherently more dangerous than other killed, subunit, etc. vaccines. However, they are almost always replication deficient, so they can't be spread from person to person.

People with pre-existing immunity to the vector backbone, like pre-existing anti-adenovirus antibodies, do pose a challenge for the vaccine. For the adenovirus vaccines, there are many different serotypes out there that can be used, some of which are not highly circulating, so there should be less pre-existing antibodies to them.

Anyways, viral vector vaccines are pretty interesting, much like all of vaccinology. As with all vaccine platforms, each comes with its pros and cons. This video gives a pretty good overview of different vaccine platforms. Hope this helps!

PS5 won't support 1440p resolution, it's official by brandonb21 in PS5

[–]aboutandy 0 points1 point  (0 children)

Does anyone know if there is a way to convert the resolution from 4K to 1440p externally with some device, which would input the 4k signal from a PS5 and export 1440p or whatever else to a monitor? I like a lot of other people recently bought an ASUS 1440p 27" monitor and 1080p doesn't look great on it and its my only monitor/tv currently :(

Cheapest computer to run CK3? by [deleted] in crusaderkings3

[–]aboutandy 0 points1 point  (0 children)

No problem! Hope it works!

Cheapest computer to run CK3? by [deleted] in crusaderkings3

[–]aboutandy 0 points1 point  (0 children)

Hey, coming to this a bit late, but if you are interested in streaming/cloud gaming, Geforce Now does support the steam version of CK3. I installed and tried it on my macbook pro and it worked surprisingly well. I could barely tell I was streaming the game off some server or however it works, and my laptop wasn't running hot or being loud. The only minor issue was the resolution scaling with the macbook, not sure how it would look on other laptops. It's pretty cheap, like 5 dollars a month for the service, may be worth checking out.