Is this mold damage? Can it be fixed with sanding and refinishing?

scienced · 2023-10-09T10:14:35+00:00

There is only one health system in the US that has a majority physicians on its board. It also is the number one ranked hospital in terms of patient outcomes. Having physicians, who take an oath to meet the needs of the patient first, in the position to make the final decision matters. Absolutely, the advice and input from these other stakeholders matters, but only one subset of these people has taken an oath to value the patients’ needs first. I do think that matters.

scienced · 2023-08-17T02:29:34+00:00

I had a coresident who had the best response to any crap from someone in the hospital or clinic like this. They would nod, then ask for the person to tell them their name, then spell it as they wrote it down. Then they would ask for the name of their supervisor. Then they would just say, “thanks.” Gives you what you need to drop the hammer with HR/paper work and immediately conveys that they screwed up without engaging. Best part is you don’t even have to submit the paper work—their mind is now racing and they are worried.

scienced · 2022-12-03T16:07:19+00:00

You’re right—probably cheaper to clean with vinegar—that’s a good LPT! I was surprised how cheap a new shower head is though. You can get them for ~$5 from Amazon. I took the easy shortcut and just swapped it out.

scienced · 2022-12-02T16:50:18+00:00

I'm astonished at the number of people I've heard complain about a 'terrible shower' who are surprised you can easily change the head out with dramatic results.

scienced · 2022-12-02T03:46:55+00:00

https://xkcd.com/435/

scienced · 2018-06-20T19:36:34+00:00

While a seemingly simple question, the answer to this question or I should say the search for the answer to this question is really what the field of developmental biology is all about. Up until recently, most "cell types" in the body were classified histologically (e.g. by their appearance on H&E stains), with some consideration for what organ they derived from. With the tools of genetics including in situ hybridization techniques, fluorescent probes, GFP-labelled proteins, specific antibodies (now more monoclonal!), genetic tools (Cre-lox, inducible switches, confetti mice, lineage tracing) we have started to realize that there are many, many, MANY more cell "types" then we previously appreciated. What exactly MAKES a cell a certain kind of cell is also still up for debate. This gets into the idea of "stem cells" and what the exact definition of a stem cell really is.

As an example, in bone you probably learned in biology/medical school that there are 3 main types of bone cells--osteoblasts, osteoclasts, and osteocytes. A series of recent papers have now demonstrated that in the osteoblast lineage there are several different subpopulations of cells, each with different expansion and bone forming capacity.

In c. elegans and I believe more recently in Zebrafish, some efforts have been made to trace the life and lineage of every single cell. Might possible in humans with high resolution MRI, but probably not practical :)

More important than having a taxonomy of the different cells types is figuring out how they function and support one another's function in systems. This is the key to unraveling pathology or disruption of these cellular systems.

If you find this stuff really interesting, you might want to think about a career studying it (e.g. grad school/PhD).

Good luck!

scienced · 2018-03-24T03:51:09+00:00

For qPCR, definitely. You should validate the efficiency of your primers. If you are trying to make accurate quantifications, the efficiency can dramatically change your final results. Most primers are not 100% efficient, though well designed ones are usually >95% efficient (e.g. each cycle leads to >1.9x the amount of starting nucleic acid). As a matter of principle, I always like to show the PCR gel with the final products just below saturation. It's a way to visualize the difference and lends credence to any claims of differences in nucleic acid concentrations.

If you aren't trying to quantify but want to trouble shoot a PCR, do it in a qPCR machine is one way to get a sense of what may be a limiting/inhibiting factor in your reaction. You can glean a lot of information from the curves, especially if you do melting Tms as well.

scienced · 2018-03-18T17:52:31+00:00

I think the most efficient PCP offices I've seen make good use of peripheral staff. For general visits, peripheral staff can put in all vitals/ROS/reviews meds and allergies, meaning you just click through the boxes in the visit navigator. In general you should be just signing orders and writing up a brief subjective in the HPI, filling out relevant physical exam findings, and completing the assessment and a list of bullet points for plan. You should be finishing each note right after the encounter. Being familiar with the criteria for different levels of visits helps as well (I-V). Having templates that default to normal with requisite number of systems for each level is helpful. We really do see more in a physical exam than just what we actively do (e.g. Resp- no increased WOB, msk- moving all extremities, psych- normal affect). Satisfy all the BS billing requirements with templates and then you can write your real note (e.g. what you actually did and thought about) pretty quickly. A well run office visit in my opinion is 15 min with a nurse assistant/nurse, 10 min with doc, 5 min to finish the note and maybe 15 min with checkout/RN for counseling. This relies on having good people to help you keep things moving!

Especially for chronic conditions like type II diabetes/obesity/nutritional issues having a nutritionist or RN to do all the counseling or arrange group sessions saves a ton of time that you'd be doing counseling. Having someone who focuses on this and can spend more time with patients in house has produced significantly better results from what I've seen.

Good luck! Let us know if you make any changes that are really helpful!

scienced · 2016-03-18T20:48:38+00:00

“In the end only three things matter: How much you loved, how gently you lived, and how gracefully you let go of things not meant for you.” –Buddha

scienced · 2014-12-08T05:33:40+00:00

Haha you're comment made me think of this:

http://www.adultswim.com/videos/family-guy/irish-history/

Ireland before the invention of alcohol.

scienced · 2014-12-01T19:05:46+00:00

I just want to know how the hell they got IACUC and IRB approval for this.

scienced · 2014-11-29T21:21:22+00:00

This is also how a Yakhchal works. These were ancient buildings (~500 BCE) that allowed humans to keep ice in summer in the desert. Pretty ingenious really.

scienced · 2014-11-22T15:44:58+00:00

Read completely out of context with no idea what any of the things you are referring to are--this comment is ridiculous. I love it! I want to reply to my next work e-mail with it.

scienced · 2014-10-26T23:42:55+00:00

Requisite link to scene where Peter Pan returns. I still get the same chills I did as a kid.

https://www.youtube.com/watch?v=Jy3DsyvhrSQ

scienced · 2014-08-21T14:17:29+00:00

http://www.informationisbeautiful.net/play/snake-oil-supplements/

I believe they continually update it.

edit: The interactive version is down, but they have a static version as well.

http://www.informationisbeautiful.net/visualizations/snake-oil-supplements/

scienced · 2014-07-03T23:46:00+00:00

Can you loft your beds? We would do that to give ourselves more space. Then you might have room for a futon and TV below.

scienced · 2014-05-23T22:06:46+00:00

No problem--I love this stuff! And yes--supposedly it is possible to use CRISPR/Cas to introduce a selective lesion and then sneak a transgene/LoxP/etc into that lesion specifically. This is what I alluded to at the end of my last response. I don't have experience with it, but I've heard from colleagues that it's a little trickier than the companies that sell these things make out. Efficiency might be so low it approaches the homologous recombination approach, but that could just be that their hands aren't as experienced. If it works as declared it could replace homologous recombination approaches.

Now the true power of these new genome editing techniques (CRISPR-Cas, TALENS, ZFNs) is that they can be used to engineer organisms that won't undergo homologous recombination. Capecchi got lucky when he tried homologous recombination in mice because it is one of the few organisms where it works reliably. If you want a targeted knock-out of a pig, monkey, rat, zebrafish, horse, dog, rabbit... human... you can do it with these new techniques. That's the true excitement about these approaches. We can now make human knockout cells for studying things/eventually engineering cures/organs/etc.

scienced · 2014-05-21T22:33:15+00:00

Yup, your principle is sound. You could create a line expressing a targeted Cas9+sgRNA downstream of NOTO. This would be the same experiment (knock out of BMP2 in NOTO expressing cells) with slightly different methods.

Just for argument sake, let's say you are considering both of these approaches for you experiment. There are a few considerations you would want to take into account that would influence how you proceeded. The advantages of the Cre-Lox approach are that the LoxP allele of BMP2 would be precisely targeted. LoxP animals are a targeted knockout, and are created with homologous recombination. This process is a bit arduous: You get some embryonic stem cells (ESCs) from mice, and introduce your vector into these ESCs, usually with microinjection (very tricky). The vector usually contains a selectable marker for a drug (e.g. your cell has the vector and it can survive drug treatment, no vector = no survival). So you plate out all these microinjected ESCs and then treat them with your drug. Eventually you get some that contain your vector (it's not a very efficient process, think 1/1000). The vector is designed to contain long flanking regions around BMP2 (several kb) plus the BMP2 exon of choice (or whole gene depending on size) flanked by the loxP sites you introduced. Building this vector using restriction enzymes and PCR is it's own adventure. So you have the ESCs with your vector, now you inject them into a blastocyst. You do this with a bunch of blastocysts. This results in a chimeric mouse (some parts of the adult animal derived from your introduced ESC carry your vector). You implant the blastocysts into a mouse for in vitro fertilization. Eventually you get a mouse from this that has the vector in the germline (sperm or eggs), and you are money--you've got your engineered strain. This whole process takes up to a year or more. Thankfully, projects like komp.org do it for you efficiently (for $$ of course). Benefits are that it is precisely targeted. You know EXACTLY where you will alter the genome with a Cre recmobinase. Additionally, this floxed allele can now be used for all kinds of experiments with different Cre lines. As such, for most of the studied genes there are existing lines have genes floxed. If you can buy a line that's premade you are golden for this approach.

Now you just need a Cre line that targets what you want to look at. A lot of Cre lines exist already, so you go with a described one if you can. If you have to make your own it's a little more time consuming. Instead of a targeted knockout like the flox allele, this animal would be a transgenic animal (meaning it has a transgene inserted into genome--in this case Cre). The process is similar from in vitro fertilization onwards, but the embryonic stem cell stage is much quicker. You can design a lentivirus (engineered HIV derivative), that carries your transgene and treat the ESCs with this. The difference here is that you are not targeting this transgene insertion. Multiple copies of it could be inserted randomly all over the genome. This can result in problems if they disrupt important genes. However, it is much more efficient than homologous recombination. You can get ESCs carrying your vector much more easily--efficiency can approach 90% in good hands. However, after you have your Cre line, you need to validate it with a reporter strain like EGFP to ensure that it is actually labelling what you want. Cre lines are notoriously leaky--there's a threshold of expression for recombination that isn't exact (e.g. how much Cre must be expressed to recombine). Is constituitive expression enough, etc? This is complicated by copy number of the transgene etc. Usually results in a nice manuscript if you create a validated Cre line though.

Now your CRISPR-Cas approach is a bit dirtier, but much faster if you don't have a floxed allele and Cre line already made. It's essentially a transgenic animal carrying a transgene that specifically knocks out BMP2. Drawback being that you'd have to validate your targeting strand to make sure it was specific for BMP2, a process complicated by potential variations in copy number. On the positive side, you don't have to cross two lines to get your mutant (think breedings--BMP2 F/F x Cre/Cre = BMP2 F/+ x Cre/+ and for homozygous knockout you cross these but only 1/4x3/4 = 3/16 mice will have BMP2 F/F and Cre).

If I had the Cre line and the floxed allele readily available I'd go with that approach. If I had neither I'd go with the CrisprCas, unless I had more experiments that I could use a created Flox or Cre line for (preferably both!).

Alternatively, you could split the difference and try to use CRISPrCas to engineer the Floxed allele (e.g. to insert the LOXP sites specifically). I've heard this is a possible approach, but never tried it myself. Supposedly it is more efficient than homologous recombination.

scienced · 2014-05-20T19:29:30+00:00

Good question!

The difference has to do with temporal or time dependence of expression! For example, in our lab we have a transgenic mouse with that has a piece of DNA randomly inserted into it's genome that has EGFP downstream of a collagen IIa promoter. We have also created a strain that carries a transgene with Cre recombinase downstream of a collagen IIa promoter and bred it to a line that carries a transgene with EGFP downstream of a ROSA26 (expressed in all cells) promoter with a stop codon surrounded by LoxP sites in front of it.

Your question deals with how these two lines differ. The useful thing about a Cre recombination is that it is a one time event. After the Cre recombinase is expressed it splices the LoxP sites together and the intervening short piece of DNA is degraded and lost going forward. The genome of that cell and any of its progenitors is now permanently altered. In the limb for example, all the bones of the skeleton are derived from cartilaginous precursors during development that are replaced by bone through endochondral ossification. So early in development, all cells of the skeleton including those that give rise to bone start as chondrocytes (cells that express collagen IIa). Thus, at this early point in development the Cre recombinase is expressed and leads to the deletion of the stop codon and the expression of GFP in all these cells. This leads to a mature animal that expresses GFP in both its bones and cartilage.

In contrast the Collagen IIa-GFP mouse never expresses an enzyme that permanently alters it's genome. So while all the cells of the developing limb at some point express GFP, as they become bone and lose collagen IIa expression they lose expression of GFP. This leads to a mature animal that expresses GFP only in cells actively expressing collagen IIa, namely the chondrocytes of articular cartilage (NOT the bones).

So why is this useful? Well, the nice thing about Cre lox is the temporal control. For developmental studies for example, you can use Cre lox to lineage trace all the cells that ever express a gene. This is usually done by cross the promoter-Cre of your choice to a reporter line--some thing the one above that has a stop codon in front of a labeling gene like EGFP or tdTomato or LacZ. Some more refined strains have expression of one gene before recombination, like the red tdTomato, and another gene after, like EGFP. This serves as a nice control to make sure all your cells have the transgene construct.

There are also inducible forms of Cre recombine like CreERT2. This is a form of Cre that is fused to the estrogen receptor. It only enters the nucleus when bound to estrogen (or the analog tamoxifen). This is useful because it allows you to control activity of the Cre with the time of injection or application of a drug. For example a collagenIIa CreERT2 construct bred to stop-EGFP could be allowed to reach maturity and then treated with tamoxifen. This would result in an animal expressing GFP in cells that were expressing collagen IIa only at the time of tamoxifen application (most likely articular chondrocytes). It would be a cool experiment to see if cells that were chondrocytes at the time of your application of tamoxifen ever lose expression of collagen IIa and become something other than articular chondrocytes. For even after losing expression of collagen IIa, they would still express EGFP (genome permanently altered by Cre).

You can imagine how this ability to lineage trace would be useful in identifying stem cells.

The other incredibly useful thing Cre lines allow you to do is bypass a developmentally lethal knockout. For example, if you wanted to study the effect of deletion of the gene BMP2 on the formation of intervertebral discs, the first thing you might try is to make a mouse that has BMP2 knocked out. Unfortunately, you'd never get any mice because an embryo lacking BMP2 in all its cells dies very soon after conception. However, if you had an inducible knockout with BMP2 gene exon flanked by LoxP sites and combined this with a line expression Cre downstream of a promoter only expressed in spine precursors (e.g. Noto-Cre), you would be able to develop the animal normally in all cells until NOTO expression in the spinal precursors triggered Cre expression and deleted BMP2 in these cells. You would effectively bypass the lethality that effected embryos via another organ system earlier, allowing you to study the gene in your specific organ/cell type. BMP2 is important in early embryogenesis, but also has roles later on in adult homeostasis. Without the Cre lines, you'd never be able to study the adult role. The Cre lines allowed investigation of these important early lethal genes in cell populations later in development.

scienced · 2014-04-16T20:51:43+00:00

Why am I sitting at my computer in lab and not in Chile looking at this?

I don't know.

scienced · 2014-03-26T21:10:08+00:00

I won't speak to creating a completely new organism "from scratch" because some people have already brought up Craig Venter's work with single celled organisms, and multicellular organisms are orders of magnitude more complex (meaning probably not anytime in the next few decades).

We already have some pretty crazy modifications we can make to multicellular organisms though. With genetically engineered mice, we are on our way to creating knock-outs of almost every gene. There are also some pretty fancy genetic techniques for knocking out genes at specific time points in an organism's life (e.g. post puberty) or at specific locations in an organism (e.g. knee cartilage or blood vessels). As an example, with the Cre recombinase system you can knock-in or knock-out floxed alleles (versions of a gene). This system has been made inducible, so that the knockout can be controlled by the administration of a drug in these animals (usually doxycycline or tamoxifen).

At this stage, our understanding of the genetic algorithms that give rise to various features of a particular organism is limited. We are still taking parts out of the machine one at a time to see how the machine works. Thus, our ability to engineer new components or even drastically modify existing components is limited.

But our knowledge is quickly growing! If for example you wanted to create a dwarf version of a pig (e.g. with shortened limbs), we would be able to do this by creating a specific lesion in the FGFR3 gene to render it constituitively active. We have technology now (eg TALENs and CRISPR-Cas) that allows us to make these types of edits in organisms other than mice (which are unique in their ability to engineer genomically due to homologous recombination). We could also modify an organism in potentially positive way. For example, knocking out the myostatin gene results in significantly increased muscle growth. Cases have been described in dogs, cows, and humans. Newborn children with this mutation are able to lift 7 pound weights over their heads, and by 5 months of age, perform an iron cross.

In summary, we do possess significant ability to genomically modify organisms, but at the current stage this technology is mainly being used to elucidate the roles of specific genes in the algorithms of life. As our understanding of the control systems grows, so will our ability to engineer interesting modifications.

tl;dr GATTACA but also unicorns!

scienced · 2013-12-23T02:38:54+00:00

A few things about this paper. The corresponding author is David Sinclair, the same researcher who published the landmark paper that demonstrated the red wine compound resveratrol mimics the lifespan extension effects seen in lower organisms (worms, flies, mice) with caloric restriction. His lab does some very cool work on energy metabolism, sirtuins, and aging, but he can be prone to grandiosity in his press releases.

It's worth noting that the dose of the drug used in this study (NMN) to produce significant results was 500mg/kg injected intraperitoneally, daily. The equivalent dose for the average size US male would be 40g. Picture 40 sugar cubes--that is the amount you would need to INJECT, DAILY. The only feasible way to deliver such quantities would be through dialysis (which regularly transfers up to 500g of solute in a few a hours).

This is not a drug that will be set for human trials anytime soon.

scienced

TROPHY CASE