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Learning from Research, The Results.

Learning from Research, The Results.

This is the part where my brain is going to explode. I might need to break this up into more than one post.

RESULTS
Preparation of HMECs

A single HMEC in its log phase was plated, and expanded to 1.4 × 106 to 1.5 × 106 cells (Fig.1). Plating efficiency during the two transfers of plates was 67 ± 0.9(mean ± SE)%. Based on these values, the number of cells that should have been produced at the time of harvest was calculated as 3.2 × 106(1.4 × 106/0.67/0.67). This value predicted that each cell harvested underwent 21.6 generations from the initial single cell. Doubling time was 48 h.

Strategy of cell culture. A single HMEC was inoculated in a well by limiting dilution, and the cell was expanded up to approximately 106 cells. Based on the plating efficiencies during the two transfers and the actual final cell count, the number of cells that should have been produced at the time of harvest and the number of generations observed were calculated. DNA was extracted from the final cells, and used for bisulfite sequencing. Six independent cultures were performed.
Slide1

HMEC – Human Mammary Epithelial Cells. They were put into a container, allowed to reproduce, and then they were checked to see if the right number of cells were made after specific numbers of generations. There were six containers of these cells. Once enough generations had passed and there were enough cells, their DNA was tested with the bisulfate test (illustrated in my earlier post.)

Gene Selection and Their Expression Levels

Methylation statuses were determined by bisulfite sequencing for CGIs in the promoter regions of the E-cadherin,p41-Arc, SIM2, 3-OST-2, and Cyclophilin A genes; CGIs in the downstream exon/introns of theE-cadherin, p41-Arc, and SIM2 genes; CpG sites outside CGIs of the E-cadherin and p41-Arcgenes; a NM-CGI of the MAGE-A3 gene; and differentially methylated region (DMR) of the H19 gene (Fig.2A). The former five genes were selected because they had CGIs in the downstream exon/introns that met a strict criterion of CGIs, regions of DNA of >500 bp with a G+C ⋝ 55%, and observed CpG/expected CpG of 0.65 (Takai and Jones 2002). The MAGE-A3 gene and the DMR of the H19 gene were selected as a representative NM-CGI and a region critically involved in genomic imprinting, respectively. By quantitative RT-PCR analysis, their expression levels were shown to range from almost none (SIM2 and MAGE-A3) to very high (E-cadherin), with p41-Arc, 3-OST-2 andCyclophilin A being intermediate (Fig. 2B).

Structures and expressions of the genes analyzed. (A) Schematic representation of the genomic regions analyzed. Regions analyzed by bisulfite sequencing are shown by closed boxes, and designations A–L correspond to panels in Fig. 3. CGI-P: a CGI in the promoter regions; CGI-outside: a CGI outside the promoter regions; Non-CGI: CpG sites outside CGIs; and DMR: differentially methylated region. All panels are drawn to the same scale. (B) Expression levels of the seven genes in HMECs.
Genome Res. 2003 May 13(5) 868-74, Figure 2

Sorry, I can’t even. All I know from this is that they looked at the results of the bisulfite sequencing and found what they were looking for – the methylation status in the CpG Islands from promoter regions of DNA stayed almost exactly the same. Unmethylated CGIs from non-promoter regions were more likely to become methylated. I’m afraid I don’t have the ability to explain this to you or tell how accurate or flawed it may be. I’m taking the researchers’ word on it. Correct me if I’m wrong.

Establishment of How to Measure MPERs

The CGI in the promoter region of the E-cadherin gene (Fig.3A), the non-CGI region of thep41-Arc gene (Fig. 3F), the CGI in the promoter region of theMAGE-A3 gene (Fig. 3K), and the DMR of the H19 gene (Fig. 3L) were found to contain two major populations of clones. The two major populations were considered to represent the methylation pattern of the two alleles in the original single cell. The methylation patterns of the two major populations were different from each other in the six cultures, which indicated that the HMECs before cloning had diverse patterns of methylation, but the patterns were relatively conserved during the culture from a single cell to approximately 106 cells. Therefore, we measured the number of errors in the methylation pattern based upon the culture from a single cell to approximately 106 cells. An MPER of a region in a culture was calculated from the number of errors in methylation pattern as described in Methods, and an average MPER of the region was calculated from the six MPERs obtained for the six cultures.

MPERS – Mammalian Protein Extraction Reagent
AlleleAn allele is one of two or more versions of a gene. An individual inherits two alleles for each gene, one from each parent. If the two alleles are the same, the individual is homozygous for that gene. If the alleles are different, the individual is heterozygous. Though the term “allele” was originally used to describe variation among genes, it now also refers to variation among non-coding DNA sequences.

So after making all those cells, they looked to see where and whether methylation status had changed.

Distribution of unmethylated and methylated CpG sites shown by bisulfite sequencing. Unmethylated and methylated CpG sites are shown by open and closed circles, respectively. (A)–(C) A CGI in the promoter region, a CGI outside the promoter region and CpG sites in non-CGIs of the E-cadherin gene. (D)-(F) A CGI in the promoter region, a CGI outside the promoter region and CpG sites in non-CGIs of the p41-Arcgene. (G), (H) A CGI in the promoter region and a CGI outside the promoter region of the SIM2 gene. (I) A CGI in the promoter region of the 3-OST-2 gene. (J) A CGI in the promoter region of the Cyclophilin A gene. (K) A CGI in the promoter region of the MAGE-A3 gene, which is normally methylated. (L) A CGI in the differentially methylated region of the H19 gene.

Here’s where they found the differences:

Genome Res. 2003 May 13(5) 868-74, Figure 3

To examine the effect of an arbitrary selection of the “original methylation pattern” in ambiguous cases, a permutation test was performed for the CGI in the E-cadherin promoter region of HMEC10. One of the clones #5–#14 (Fig. 3A) was hypothesized as one of the original methylation pattern, and the number of errors in the methylation pattern was calculated. The numbers ranged from 18–22, and these values were expected to result in the average MPER ranging from 0.022–0.023. Similar permutation tests were performed for the CGI in exon 2 of the E-cadherin gene of HMEC12 and HMEC15. The numbers of errors in methylation pattern ranged from 13–16 for HMEC12 and from 12–15 for HMEC15, and these values were expected to result in the average MPER ranging from 0.050–0.058. These showed that arbitrary selection of the original methylation pattern in ambiguous cases does not seriously affect the resultant average MPER.

Some changes weren’t so cut and dried, so they checked those cases and found that they weren’t significant enough to change the findings.

The efficiency of bisulfite conversion was examined by analyzing DNA with no methylation in the CGIs in the promoter region and exon 2 of the E-cadherin gene. In the CGI in the promoter region, none of the 600 cytosines at CpG sites (30 CpG sites per clone, 20 clones analyzed) remained unconverted, showing that unconversion rate was almost 0 in this region under our experimental condition. In the CGI in exon 2, one of 483 cytosines at CpG sites (23 CpG sites per clone, 21 clones analyzed) remained unconverted, showing that the unconversion rate was 0.0021. These values showed that the MPERs in CGIs in the promoter regions are 10-fold more than the unconversion rates.

The bisulfate conversion was also tested separately for control to make sure the results would be valid in the experiment. This reinforced the finding that the promoter regions stayed stable.

MPERs and Fidelities of Methylation Pattern in the Genome

The average MPERs obtained for each region are summarized in Table1. Unmethylated CGIs in the promoter regions showed MPERs between 0.018 and 0.032 errors/site/21.6 generations. In contrast, CGIs outside promoter regions showed significantly higher MPERs, ranging from 0.037 to 0.091 (P < 0.01 or 0.005). MPERs in the CGIs outside the promoter regions were more than twice as high as those in the promoter regions of the same genes. MPERs in Various Genomic Regions

NM-CGI of the MAGE-A3 gene and methylated alleles of the DMR of the H19 gene showed MPERs of 0.002 and 0.007, respectively. Any genomic regions that were normally methylated, whether or not they were in CGIs, showed significantly lower MPERs than those unmethylated. This was particularly clear when the MPER of the allele methylated at DMR of the H19 gene was compared with that of the other unmethylated allele.

Interpretation of the tables, summary of findings.

This is not as good as part one, sorry. In other news, I couldn’t watch Besharam because it sucked, so I didn’t learn any Hindi, either. One more post to go in this series. Anyone who can clarify/explain better than I can, please comment – I’d appreciate it.

Science Education – How I Would Do It.

Science Education – How I Would Do It.


Of course, this is assuming that the world was a sensible place and I was in charge of all the important decision-making. Heh.

Over time, I’ve come to realize that a lot of the things I was taught in school didn’t stick because they weren’t interesting. They weren’t interesting because they were unrelated to my life, and I couldn’t see how they could possibly be important to me. I memorized things for tests, and I did a darn good job of it, good grades, good standardized test scores, but only because I had to, not because I wanted to.

As I got older some of it came back – and it stuck better because I had context to put it in. Before kids and before antidepressants, I read a lot of romance novels for escape (I know. . .I’m not proud, but I had an excuse.) Soon I discovered that there was a sub-genre of Historical Fiction – and some of these authors were real history buffs who included a lot of factual information. In the context of a story, with characters and plots that engaged me, I was finally learning something about history, which had bored me to tears in High School.

Later, I started reading some of the books and papers that had been assigned back then. . .suddenly they were interesting and made sense – because I now had a context for them. The context continued to expand, and more information became part of what I knew.

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For me, possibly moreso than for many people, context is essential. My ADHD mental filing system demands context and associations not only for learning, but for retrieving that learning. So when I teach people what I know, I teach it in context. I learn a lot by making mistakes, so I teach “do it this way because this other way doesn’t work,” and “we do it this way because otherwise we break this piece and the whole thing is ruined.” I teach “This part seems boring, but here are all the cool things we can do with it later.”

I also learned a lot from raising my own kids and volunteering in their schools, helping all kinds of other kids learn. You need to be able to express a single piece of information many different ways in order to get different kids to understand it. As a volunteer, I was able to sit with individual children and small groups. The kids who didn’t understand things when they were taught the same way to all 30-something students would get it if I spent some time with them and figured out what their individual contexts were.

_______________

Fast forward to the mid 90s – I started antidepressants, and then I discovered that my ADHD had not actually gone away as the experts had told my parents it would, and as my parents told me it had. Now I had a reason to learn about the brain, starting with disorders and injuries, and what they taught us about the functions of various structures. That gave me a context to learn about brain development and genetics. This led to investigating epigenetics. Along the way, it also tied in to reading medical and science blogs and books, and any time a piece of knowledge stuck to something that was relevant to something I already knew, it also became relevant.

So why do you want to listen to someone who doesn’t have a degree in science or medicine when it comes to science or medicine? Because of the way I’m learning it. That whole “Translating Science into English” thing I mentioned a few posts back. Scientists have their own language, and it’s important that they do so they speak with clarity and precision. But if you don’t have the context that they do, it’s hard to understand – and easy to misinterpret. I didn’t learn this in the linear fashion that they did.

If you were to teach me vocabulary and facts and mechanisms, I’d remember it just as well as I did in high school. But give me a study of something that relates to something that interests me, and I will look up all those words and facts and mechanisms, and they’ll make sense because they’re part of something else. They have more meaning when they’re in context.

The other thing I learned came from watching scientists argue with one another. While they’re not always polite, they always present evidence. Most of them are critical thinkers, when someone says something that is questionable, they will (sometimes very methodically and in great detail) explain the flaws in the reasoning. Following along with this taught me the scientific method and why it’s important, how to evaluate how robust the data is by looking at the size of the study, the quality of the blinding, the strength of the variables and controls, how well it integrates existing evidence (and how strong that evidence is) and, most importantly, no matter how good a study may be, it’s never PROOF. It also doesn’t prove other things that weren’t part of the study. It’s also probably not a major breakthrough.

I learned about p-values, journal impact factors, the good and bad of peer review, the pros and cons of open access. I learned that not all “evidence” is actually evidence.

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The problem that many, many scientists have, though, is that they forget what it’s like to not know this. Sometimes they present what they know in a way that is off-putting to laypeople. Sometimes they present a press-release version of their findings, breathless with excitement and full of hyperbole, and that’s even worse. (That’s what we have The Daily Mail and Huffington Post for. Let them do their job.)

So if I were a science teacher, or I were designing a science education program, I’d throw out teaching the basics as freestanding facts. Get rid of the rote learning. Give the students just enough information to dive into a challenge and figure out the rest. Give the kindergarteners a bowl of cream and some food coloring and dish soap – let them play and then tell them how it works. Let the older kids listen to each others’ heartbeats, check each others’ blood pressure, draw pictures of hearts and veins and arteries, and use that to introduce the circulatory system. Make everything part of an experiment that related directly to them so that it was important. Let them figure out what’s correct and what’s incorrect as much as you can on their own by giving them questions as much as answers. Make the science interesting and integrate critical thinking into the lessons, and get them excited. This will be good for them, and good for society, because they’ll question everything – and come up with their answers based on what evidence is best supported.

Learning from Research, Slowly and Methodically.

Learning from Research, Slowly and Methodically.

I was given a challenge on Twitter, and some people dismissed me as a failure because I didn’t have the academic background to come back with a quick answer. (I also discovered that I knew the answer, but forgot the words because of post-surgical anomia. I digress.) I find that this is a problem with a lot of people with certain types of expertise. They forget what it was like back when they were first learning, and no longer have the patience to explain. I don’t think it helps that there is a shit-ton of people on the internet spouting nonsense and being taken seriously. Naturally, some of them will assume that I’m doing the same, but I really don’t want to be lumped in with them, so I’m going to show them the process I go through, and how seriously I take learning new things and separating fact from fiction.

As I said in my previous post, you guys are wicked smart, and I am very often in awe of how much you know. But one thing you’re not so good at is communicating to people outside your fields of expertise. This is why we have bad science journalism. Ask Ed Yong. However, if you want to stop all your discoveries from degenerating into misrepresentation or woo, then you need people who can translate Science into English.

I was given a long, information-dense study, Fidelity of the Methylation Pattern and Its Variation in the Genome by Malcolm M. Campbell, so it’s going to take several posts to dissect, research, learn the background information, and try to explain it in an accessible way. I fully expect to be wrong several times, and encourage people to correct me – in such a way that ordinary people can “get it.” So here goes:

Abstract

The methylated or unmethylated status of a CpG site is copied faithfully from parental DNA to daughter DNA, and functions as a cellular memory. However, no information is available for the fidelity of methylation pattern in unmethylated CpG islands (CGIs) or its variation in the genome. Here, we determined the methylation status of each CpG site on each DNA molecule obtained from clonal populations of normal human mammary epithelial cells.

Methylation turns genes or pieces of genes “on” or “off”. There’s a detailed explanation of various ways it does this in the components of the whole process from DNA to cell, but it’s kind of hard to understand if you haven’t done a lot of reading beforehand. I’ll give you the link anyway.

CpG sites – the quick and dirty Wikipedia definition is this: The CpG sites or CG sites are regions of DNA where a cytosine nucleotide occurs next to a guanine nucleotide in the linear sequence of bases along its length. If you don’t remember from your Biology classes, or your biology classes never taught you, your entire DNA strand consists of combinations of four nucleotides – Cytosine, Guanine, Taurine, and Adenosine. I’m not going to get into that right now, because it’s just going to confound this with too much information, but if you think about the movie “Gattaca,” you’ll notice those four letters. In a movie about genetic engineering. Because those are the four letters you see in an illustration of a piece of DNA. The researchers were looking at the parts where the cytosine and guanine were next to each other.

Specifically, they were looking at epithelial cells from normal breast tissue. The link may be a little difficult to understand, but I think if you read all the way through, you’ll at least understand some of the reasons these cells were chosen. They have a lot of unique characteristics, and they’re pretty tough.

So the idea here is that we already know that if the cytosine and guanine pair are methylated in the on position or the off position in the DNA, that they’re going to stay that way in the cells that are produced by those instructions from the DNA. What we don’t know is that if that pair is unmethylated, will the cells made from the DNA instructions also be unmethylated? IOW, if they’re not already told to be switched on or told to be switched off, will they still be in that “neutral” position? In order to test that, they took a bunch of those epithelial cells and tested each one to see if it was methylated or unmethylated so they could get them to reproduce and see what happened.

This illustration is not specific to this piece of research, but keep reading, and you’ll see how it relates.. I wanted to give you a visual aid in case you learn better that way.

Methylation pattern error rates (MPERs) were calculated based upon the deviation from the methylation patterns that should be obtained if the cells had 100% fidelity in replicating the methylation pattern. Unmethylated CGIs in the promoter regions of five genes showed MPERs of 0.018–0.032 errors/site/21.6 generations, and the fidelity of methylation pattern was calculated as 99.85%–99.92%/site/generation. In contrast, unmethylated CGIs outside the promoter regions showed MPERs more than twice as high (P < 0.01). Methylated regions, including a CGI in theMAGE-A3 promoter and DMR of the H19 gene, showed much lower MPERs than unmethylated CGIs. These showed that errors in methylation pattern were mainly due to de novo methylations in unmethylated regions. The differential MPERs even among unmethylated CGIs indicated that a promoter-specific protection mechanism(s) from de novo methylation was present.

This explains how they figured a reasonable range of variation. The “islands” of unmethylated cytosine/guanine pairs in five genes over 21.6 generations (this is statistics, not absolute numbers. You clone enough cells, you sure as heck can get six tenths of a generation.) stayed unmethylated most of the time. This came from promoter regions, which are the areas in DNA that call the shots. It’s more likely that instructions from promoter regions are going to be followed.

The unmethylated cells that didn’t come from promoter regions showed more deviations – the cells after several generations were twice as likely to be different from the originals than the ones that came from the promoter regions. The methylated cells, which, as I mentioned, already have the specific instructions to turn a gene on or off, were more likely to maintain their integrity even if they weren’t from promoter regions. The unmethylated cells didn’t’ have that instruction, and hadn’t been told to stay unmethylated (because they weren’t from promoter regions) and so they just did whatever seemed right at the time and, well, mistakes were made.

CpG methylation is known to serve as cellular memory, and is involved in various biological processes, such as tissue-specific gene expression, genomic imprinting, and X chromosome inactivation (Jones and Takai 2001; Bird 2002; Futscher et al. 2002;Strichman-Almashanu et al. 2002). These important functions of methylations are based upon the fact that the methylated or unmethylated status of a CpG site is faithfully inherited. The methylated status of a CpG site is inherited upon DNA replication by the function of maintenance methylase, represented by DNA methyltransferase 1, which is located at replication forks and methylates hemimethylated CpG sites into fully methylated CpG sites (Leonhardt et al. 1992; Araujo et al. 1998; Hsu et al. 1999). The unmethylated status of a CpG site is inherited by not being methylated upon DNA replication or any other occasions. Unmethylated CpG sites generally cluster to form a CpG island (CGI), and most CGIs are kept unmethylated (Gardiner-Garden and Frommer 1987; Bird 2002). Methylations of CGIs in promoter regions are known to cause transcriptional silencing of their downstream genes by changing chromatin structures and blocking transcription initiation (Bird 2002;Richards and Elgin 2002). There are limited numbers of CGIs that are normally methylated (normally methylated CpG islands; NM-CGIs) (De Smet et al. 1999; Futscher et al. 2002). CpG sites outside CGIs, especially those in repetitive sequences, are also normally methylated (Bird 2002).

CpG methylation is important. It is carried on pretty faithfully when cells reproduce. It’s also important that unmethylated CpG remains unmethylated, and that’s usually passed on to new cells as well. Most of the unmethylated sites form a cluster called a CpG Island, or CGI. If these unmethylated CGIs become methylated, then it changes what genetic instructions get turned on or off in future generations of cells, if they’re in promoter regions. But it’s not always bad for CGIs to be methylated, because sometimes that’s on purpose.

I’m going to hold off on the transcription and chromatin stuff for later, because I think it’ll stick better when the paper goes into more detail.

To keep the methylation pattern, maintenance of both methylated and unmethylated statuses of CpG sites during DNA replication is necessary. However, the fidelity of the methylation pattern has been analyzed only for the maintenance of the methylated status (Wigler et al. 1981; Otto and Walbot 1990; Pfeifer et al. 1990). The fidelity in maintaining the methylated status of an exogenously introduced DNA was shown to be 94% per generation per site by Southern blot analysis (Wigler et al. 1981). The fidelity in maintaining the methylated status of a CGI in the 5′ region of the PGK1 gene, which was derived from the inactive X chromosome, was estimated to be 98.8%–99.9% per site per generation by the ligation-mediated PCR method after chemical cleavage of DNA (Pfeifer et al. 1990).


We’ve already studied methylated CpG sites and found that it’s pretty consistent. Some studies attesting to that are cited. We know that keeping them unmethylated is also important, but that hasn’t been investigated to our satisfaction.

Normally unmethylated regions might show different fidelities from normally methylated regions. Even among the unmethylated CGIs, the fidelities of their methylation pattern have been suggested to be different according to their location against a gene promoter. Methylation of CGIs in promoter regions almost always leads to transcriptional silencing while that of CGIs outside promoter regions does not (Gonzalgo et al. 1998; Jones 1999). Considering the cellular expense in maintaining methylation pattern, a cell could sacrifice the fidelity of methylation pattern for CGIs outside promoter regions. In addition, by recent genomic scanning techniques for methylation changes (Ushijima et al. 1997; Toyota et al. 1999; Costello et al. 2000; Jones and Baylin 2002), aberrant methylations of CGIs in cancers are observed in a nonrandom manner (Toyota et al. 1999; Costello et al. 2000; Kaneda et al. 2002a; Kaneda et al. 2002b). It is indicated that CGIs outside promoter regions were more frequently methylated than those in promoter regions (Nguyen et al. 2001; Takai et al. 2001; Kaneda et al. 2002a; Asada et al. 2003).

Unmethylated CGIs are more likely to change than methylated ones. Unmethylated CGIs from promoter regions of the DNA pretty consistently shut down the things they’re supposed to shut down, exactly as planned. Unmethylated CGIs from outside promoter regions of the DNA are not so good at that – they’re more likely to become methylated when they’re supposed to stay unmethylated. Some of this methylation of unmethylated CGIs has been seen in cancer. So that’s one example of why we don’t want this to happen.

Here, we analyzed the methylation status of each CpG site on each DNA molecule by the bisulfite sequencing technique (Clark et al. 1994) in six clonal populations of normal human mammary epithelial cells (HMECs), for CGIs in the promoter regions, CGIs outside the promoter regions, and CpG sites outside CGIs. By analyzing the deviation from the most common two patterns, MPERs, which reflected the fidelity in replicating both methylated and unmethylated statuses, were measured.

Like a five-paragraph essay here. Restating what they’re going to do and how they’re going to do it. Remember the illustration? Bisulfite sequencing technique. (Really detailed explanation, Wikipedia explanation).

And now my brain is very, very tired. I am going to watch “Besharam” because I’m also trying to learn Hindi, and I might as well be looking at Ranbir Kapoor while I’m doing it. Heh. I will continue this in a later post. Feedback is welcome and encouraged.

Science People!

Science People!

I’ve been getting a lot of attention on Twitter for the last couple of posts, and that’s given me a lot of articles to read, blogs to keep up with, and Twitter users to follow. Some people got a little testy, and I don’t blame them, because they know more than I do. I get it.

Let me tell you something right now. I am not a professional scientist. I got my Bachelor’s degree in Spanish Language and Literature back in the early 80s, and distanced myself from science since I had to take my only B-track class in all of High School in Biology. I didn’t get it, I didn’t see the point, I put no effort in, and I sucked at it.

That’s ADHD.

But then I started reading books about the brain, and that struck a chord with me because my brain is not the nice neurotypical model. I started reading blogs and websites about the brain, and medicine, and genetics. I learned how to read published research (and occasionally got friends who would sneak me links to full text articles) and would search in the middle of searches when I found terms I didn’t understand or biological processes or mechanisms that were new to me but essential to understanding what I was reading.

This obsessive pursuit of information is also ADHD, BTW.

This means that there are gaps in my knowledge. I am not ashamed to admit that you know more than I do. Please don’t get angry with me when I’m wrong – explain to me why I’m wrong and then tell me how to understand it the right way. I don’t want to be right to win arguments or lord it over people, I want to be right because I have the correct information. You can help me with that.

Thing is, one thing I know I’m really good at is teaching other people things. I take my mistakes, the process by which I figured something out, and the way it works at the most basic level, and try to use that to explain what I know in a way so that other people can “get it.” There are several college students out there pursuing degrees in science because I got them all excited about it. They’re getting the chance I missed out on.

So, you want more minions? (MUHAHAHAHA!!) Give me comments. Help me understand. Because if you help me understand, I can help other people understand. I’m an intelligent woman, I’ll get it pretty quickly, and when I don’t, I’m not in the least ashamed to admit that I was wrong. We can have a mutually supportive and respectful interchange, and I’ll do my part to explain things in an accessible way, using the tools you give me.

Really. Comment. email. Bring it on. I love you guys!

Epigenetics – I do not think that word means what you think it does.

Epigenetics – I do not think that word means what you think it does.

And I kind of have a bone to pick with Scientists who are actually contributing to the problem. Epigenetics is an essential biological process that takes place at the molecular level. Each one of the hundred trillion or so cells in the human body was created via the epigenetic process. Nothing has to magically happen. All you need is cells, food for the cells (usually glucose, yum!) and DNA.

Unfortunately, the amazing and fascinating research into epigenetics has led to a description of epigenetics as “genes plus environment.” If you are a scientist, or even understand science, you recognize that this does not mean that some sort of environmental factor from outside the body is necessary for the epigenetic process to take place. But if you’re a layperson, that’s exactly what you might think when you hear that. In fact, for quite some time I’ve been debating with a couple of people who believe in this magical concept of epigenetics, and you scientists (whom I otherwise love dearly) are just not helping!

The agouti mouse study that showed a change in coat color (linked along with other references in this previous post) was really exciting, and the public glommed onto it because there was the evidence, right in front of their eyes. In no time at all, alt-med proponents and the general public were certain that this was the answer to everything that was wrong with us. It was a great boon for supplement manufacturers, diet book writers, food conspiracy theorists, and anyone who was looking for something to blame for what was wrong with them (or society, but usually themselves.) I mean, clearly if what a mother mouse ate changed the color of her babies’ fur, then what horrible things are all these toxins doing to our genes?!?!

The thought seems to be that epigenetics is a highly unstable process that actually depends upon the correct “environment” in order to occur, and that even an unpleasant event in childhood can somehow upset it and result in a dramatic condition that can be passed down to one’s offspring. Once a person has gotten this idea into his head, it is darn nigh impossible to get it out. Homeopathic amounts of a “toxin” can have traumatic results, even worse than actual poisoning from that substance, because epigenetics. Psychiatric and neurological conditions are inflicted upon perfectly healthy infants by insufficient parental attachment or attunement. Everything is caused by environmental disruption of the epigenetic process, and everything in the environment messes up epigenetics.

Look, the reality is that what epigenetics does is take the information that’s been put into the RNA from the DNA, turn on the genes that are needed and turns off the ones that aren’t, then sends proteins off with the instructions to make new cells. At conception, when there are only a few cells, there’s not a lot of differentiation, but as fetal development continues, these instructions become more specific. “Make fingers.” “Make retinas.” “Make heart valves.” Stuff like that. During growth, the instructions are more like “make more of these cells.” During adolescence, it’s “make these a little different.” As we age, it’s “make another one just like this,” and “eh, what was that, sonny?”

The environment comes in because it is the epigenetic process during which an environmental factor can possibly alter the process, turning a genetic instruction on that should have been off or vice versa. It’s quite likely that this is what triggers many cancers that are strongly associated with exposure to a particular substance. But the possibility that exposure can impact gene expression is not the same as the inevitability of exposure altering gene expression. And this, people, is a big problem. Scientists, please think about this when you talk about epigenetics. Non-scientists, I’m going to put an explanation of how this works in the simplest terms I can come up with in another post.

Your Inner Fish

Your Inner Fish

I loved this book, and now PBS is making a miniseries with Neil Shubin. I can’t wait.

A long time ago, right after I read it, I put up a series of posts on a forum detailing the wonderful things I had learned from it. After a while, the threads were hijacked by people who just didn’t get it – or didn’t want to get it – and they disappeared into obscurity. But I stand by what I wrote, and now that this book is back in public view, I want to share these thoughts again. This is a long read, over 4,000 words, and it’s taken from a forum thread, so there are parts that don’t flow entirely well, but I don’t want to edit or rewrite it because it captures the wonder and excitement I felt when I first read the book and I don’t want to change that.

So settle down with a nice cup of tea if you’re ready to go below the fold.

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ADHD and Pharmaceutical Fearmongering.

ADHD and Pharmaceutical Fearmongering.

It’s never difficult to find articles about how ADHD is some trumped-up condition made up to excuse poor behavior and/or line the pockets of the medical industry. Whether the writer assumes one or both of these, it’s necessarily bound together with denial, ignorance, and hyperbolic claims. Sometimes all you can do is get angry, but other times the writer gives you a chance to deconstruct his points. The Price of ADHD Business is that second kind.

He opens with this blockbuster:

Over 12 million children and young adults consume ADHD stimulant and psychiatric medications in the United States. Pharmaceutical corporations generated near 9 billion dollars in 2012 for ADHD stimulant drug sales, representing 5x the 1.7 billion in sales ten years ago.

Shocking, isn’t it? Except that in the US, the Pharmaceutical Industry makes about $345 billion a year. That means that psychiatric medications make up a whopping 2.6% of the bottom line. Hardly one of their biggest players, compared to drugs for cholesterol, pain management, and cancer treatment, which are much better performers when it comes to percentage of business. Also, notice the subtle slide from “ADHD stimulant and psychiatric medications” to “ADHD stimulant drug sales,” because this will be important.

More alarming, this rate of consumption represents 3x the world’s children combined, according to data collected by Scientific American. The business model of behaviorally assessing and prematurely medicating young school age children with powerful stimulant and psychoactive drug therapy for over 40 years is now under fire. The Government Accountability Office (GAO) Child Foster Care Drug Audit Report uncovered dangerous and unethical prescribing practices. Widespread abuses of overmedicating young foster care children with ADHD stimulant as well as psychiatric medications prior to ruling out nutritional, physiological, and environmental risk factors were uncovered by the largest child foster care prescription drug audit in American history.

Of course, being a first-world country, it’s more likely that we have 3x the children being treated for, say, cancer or juvenile diabetes, or any number of other childhood diseases, so there’s something of a leap from claiming that 3x the children being treated means that something is being treated too much – or “prematurely.” I notice also that the source of the alarmist rhetoric comes from a study of children in foster care. Well, this is a problem with foster care, not with all children. Foster care children are more likely to have disabilities, both physical and mental, meaning they’re more likely to actually need treatment. They’re also covered by state medical programs that make it pretty easy to get treatment that a self-payer parent might not be able to obtain. There’s more, but if a mere scratch on the surface can reveal that we’re comparing apples to oranges, there’s not much need to go even deeper.

In today’s America parents, educators, and prominent healthcare professionals challenge the 40-year ADHD business model, as the ADHD diagnosis rate surpasses epidemic status in 2014. The symptoms of ADHD are real and in many cases can be debilitating to children as well as adults. Especially in the young child population, the ADHD business model of assessment and treatment requires immediate reform. Children have a right to receive comprehensive bio-assessments as well as behavioral assessments to determine cause of their symptoms prior to powerful stimulant and psychoactive drug therapy.

Yes, we have a serious epidemic of about 5-8% of the population. That’s massive. Not. Notice how he snuck in the disclaimer (like, some of my best friends have ADHD!!!) but still calls the diagnosis and treatment of ADHD a “Business Model.” I sense a broken irony meter. Also, have you ever taken any of these medications? The stimulants are among the least powerful ones out there, with the most immediate effect (no two-month waiting period) and little to no withdrawal problems. Lumping them in with all psychoactive medications is disingenuous, especially for someone banking on his rep as a Pharmacist.

The Diagnostic and Statistical Manual for Mental disorders (DSM) lists ADHD as a mental disorder. The DSM diagnosing criteria, created by psychiatrists, involves a subjective behavioral assessment process which forces children primarily into premature drug therapy. Although seven out of ten children may exhibit an initial positive behavioral response to stimulant drug therapy for focus and attention, the long term side effects are now known.

Yes, they are, and they’re not terribly scary. Children who start using methylphenidate or dextroamphetamine medications may grow up to be as much as one centimeter shorter than their peers. Of course, they’ll also grow up happier and more successful and less likely to abuse drugs than their non-medicated ADHD peers, but that runs counter to the narrative here.

The Johns Hopkins Child Center Study results of 2013 prove that stimulant drug therapy should not be the primary intervention in young children. This study followed four year old preschool children who were diagnosed by their physicians for ADHD and medicated with stimulant drug therapy for a six year period. When the ADHD assessments were reviewed at age ten, over ninety percent of the children were worse off in their condition. Long term side effects of ADHD stimulants may include anxiety, minor depression, as well as aggressive behavior. Additionally, the Hopkins study determined that ADHD causes an economic burden to the US exceeding 45 billion dollars, annually.

To the first sentence I say, “Well, duh.” The primary intervention should be behavioral, with medications added to supplement as needed. This is not news, and it does not run counter to what any Medical Association is recommending, even the American Psychiatric Association, which says “Behavioral therapy and medication can improve the symptoms ofADHD. Studies have found that a combination of behavioral therapy and medication works best for most patients.” Add to this yet another lie, because what the Johns Hopkins study revealed was (prepare to be shocked) that ADHD doesn’t go away, and medications don’t cure it, just relieve the symptoms while they are in effect. And the economic burden he’s talking about? That relates to the consequences of untreated ADHD – people in jail, people who are substance abusers, people who are unable to work, people who have other health issues that are related to ADHD.

Many parents are not aware that a diagnosis of ADHD for their child is a diagnosis for mental disease in accordance to the DSM. Once a young child is placed on ADHD stimulants including Adderall or Ritalin prior to ruling out causative risk factors, there is an increased health risk. Additional medications for the treatment of long term side effects may be required due to the development of other behavioral symptoms.

Actually, parents know this, because lots of the evaluations are related to getting assessments for school. And it’s not the stimulants that increase the risk of further diagnoses and additional medications as much as the fact that ADHD is usually not alone, and the co-morbid conditions are discovered because the children are being observed and treated by doctors. The medications don’t produce these problems.

For example, the GAO drug audit uncovered a 2,200 percent increase in drug expenditures for atypical antipsychotic medication reimbursement to the state of Michigan during an eight year period from 2000 to 2008. Children in foster care, as the report states, were abusively prescribed powerful antipsychotic medications including Abilify, Zyprexa, Seroquel, Geodon and Risperdal. The Michigan Medicaid system was billed an increase of 40 million dollars during an eight year period just for this one class of medications in foster care children. US Senator Thomas Carper, requestor of the GAO drug audit and chairman of the Homeland Security & Government Affairs Committee, stated “I was almost despondent to believe that the kids under the age of one, babies under age one, were receiving this kind of medication”.

Remember what I said about the creep from the “all psychiatric medications” to “ADHD stimulant medications”? Here we go with another false equivalency. Antipsychotic medications are the last resort, used for treating not just ADHD, but ADHD with serious comorbids that would make the children a danger to themselves or others. Also, remember that this is the foster children, not all children with ADHD as a whole. AND keep in mind that several of these antipsychotics are essential for the treatment of schizophrenia and seizure disorders, which are probably too legitimate to mention in the context of this article. So the figures on antipsychotics for foster children in one state is cherry-picked data that in no way reflects that there is an epidemic of children with ADHD receiving inappropriate medications.

Should ADHD be labeled a mental disease especially in young children who have not been given the right to find the cause of their symptoms prior to stimulant drug therapy? Or, should ADHD be classified as a symptom of condition with underlying causative nutritional, physiological, and environmental risk factors?

Um, yeah, it should. Get inside our heads, mister – it’s definitely a mental disease. It’s certainly not something we can choose or turn off at will. And the cause of their symptoms is mental – the other “causative” factors have been thoroughly debunked as “causes” in study after study. So this is a giant flaming strawman.

The German magazine, Der Spiegel, quoted a prominent American ADHD psychiatrist in their February 2, 2012 issue. Dr. Leon Eisenberg, who coined the term ADHD over forty years ago, stated “ADHD is a prime example of a fictitious disease.” At age 87, this was Dr. Eisenberg’s last interview prior to his death. During the last forty years, he was involved in pharmaceutical trials, research, teaching, as well as the development of social policy pertaining to child psychiatry. He was a recipient of the Ruane Prize for Child and Adolescent Psychiatry Research. Currently, over fifty percent of psychiatrists on the DSM panel responsible for ADHD diagnosing and treatment protocols have direct business ties to drug manufacturing corporations.

All that education, and Mr. Granett doesn’t know how to check snopes. How sad. As to that second claim, well, a link would be nice, but I’m not surprised it’s absent, since the actual ties would be openly disclosed and not as incriminating as the author would like them to be.

Dr. Thomas Insel, Director of National Institute of Mental Health, stated on April 29, 2013 “patients with behavioral conditions deserve better… the current assessment process lacks validity.” He supports research that better treats and may even prevent the development of behavioral symptoms in children.”

Again with a diversionary link. Psych Central has some validity, but “The Verge”? Really? Why not link to the NIH’s Research Domain Criteria which explains that what this means is that the NIH wants to have research focused on multidimensional approaches to research, and research that is targeted towards evaluation of symptoms and behaviors rather than whole conditions, because we now know that there’s a lot of crossover and and a narrower approach will produce more successful and useful research. Oh, but that would not support the POV of the author. That’s OK, now you can see what Insel was really talking about. You’re welcome.

ADHD symptoms can be reversed through a process of differential diagnosing. The elimination of nutritional, physiological and environmental risk factors prior to premature drug therapy is the new ADHD Business Model for helping children and adults reclaim their behavioral and mental health. The Action Plan for Childhood Behavioral Conditions discussed in the book Over Medicating Our Youth as well as the upcoming 2nd edition T he American Epidemic: Solutions for Over Medicating Our Youth provides critical bio-assessment information to find the cause of ADHD symptoms. This action plan provides an informational template to unite parents, teachers as well as all healthcare professionals for the purpose of helping children win the battle against behavioral challenges.

Many assessments help determine the cause of ADHD symptoms. Learn how bio-assessments for reactive hypoglycemia, diabetes, the brain-gut connection, cervical spinal alignment, exercise, whole food nutrition, brainwave optimization, and nutritional enzyme supplementation may reverse ADHD symptoms.

And now, ladies and gentlemen, we begin our final descent into woo. ADHD symptoms cannot be reversed by any of these things, and. . .OMG, this whole thing is an advertisement for. . .wait for it. . .a book co-authored by Frank J. Granett! It’s so good that he had to cite himself! And real medical terms weren’t sufficient, so we have to make up some that sound really sciency, like “differential diagnosing,” and “reclaim their behavioral and mental health,” and “bio-assesment” so we can sell ineffective treatments to gullible patients. Blood sugar problems can be diagnosed and treated without ADHD medications. The “brain-gut connection” has no supportive research except in patients with full-blown Celiac Disease. Cervical Spinal Alignment is Chiropractic’s uglier younger brother, even less useful than regular Chiropractic for treating anything, much less neurological conditions. “Brainwave Optimization,” don’t even get me started. As for the rest, we already touched on how none of these things are causative, so they are not going to be curative.

But this is what it usually comes down to, isn’t it? The voices that protest the loudest that ADHD is a fake disease created to make money by the pharmaceutical industries tend to end up thinking that it’s real enough to be treated by whatever they themselves are selling. If only there were an all-natural cure for hypocrisy. . .

Something that Breaks my Heart

Something that Breaks my Heart

Many years ago, I joined an online forum community. One of the areas on this community was for scientific discussions. I had never had much interest in science in general, but I had been reading books about the brain – partly out of curiosity about my own issues, and partly because they were really fascinating. By the time I found this place, I had graduated to reading a fair number of skeptical and science blogs. My curiosity had been aroused. At the same time, my critical thinking skills were being tested and honed. Finding a place where people were discussing research and providing evidence and links to studies about brain things that were relevant to me was wonderful and exciting.

When people posted things that were well-supported, I found new sources of information in links and searches. When people considered implications or possibilities, they provided evidence and reason for their ideas. When people posted things that looked suspicious, I went off in search of the truth, and found out all kinds of knowledge that either rebutted or supported these things.

In fact, if it hadn’t been for someone posting about epigenetics in a way that sounded an awful lot like magical thinking, I would never have delved as deeply into this fascinating process as I have, and would not have learned most of the amazing things I now know about genetics, evolution, and developmental neurology. I had learned a lot about the physical structures of the brain and their functions, but without the input of the other curious and intelligent people there, I wouldn’t have known about the interconnectivity and the complex chemical and electrical communication that makes these structures function as a whole.

This was different from reading scientific information from scientists. While some were writing for public understanding, most were writing for their peers. These were not always places to pose elementary questions or ask if some speculation you had might have some factual support. This forum was, and I looked forward to visiting it every day. The dialogues were lively, and disagreements were usually battles of who had the most robust evidence. I was interested in science, but it was this particular place that got me excited about it.

No more.

Sadly, an extremely small number of people have been given carte blanche to ignore all the rules that used to make this discussion area work. Should anyone dare to post anything remotely resembling fascinating new information, they descend upon the conversation and shut it down. Every thread looks like a copy-paste of every other thread; evidence is disregarded in favor of dogma; anyone who disagrees with what is basically the only topic of the entire area is told they are wrong with a thinly veiled insult to their intelligence.

Cognitive dissonance should be a call to arms – go out and find the truth! It should prepare you to learn more and change your mind if you discover that the best evidence contradicts what you think you know. Now, the ability to ignore that conflict is treated as the highest of virtues.

Here was a place filled with creative, curious people, who had unusual approaches to connecting ideas, different ways of putting things together and taking them apart. Here was a place where the goal was not to be right for the sake of being right, but to be right because you could show your work. Here was a place that was exciting and interesting and challenging.

It is gone, and I don’t anticipate it ever coming back. I used to direct people to this place to get information; now I tell them they won’t find what they’re looking for there. It is hostile and uninviting to the very type of thinkers it used to attract. I miss it terribly.

What Does Random Mean?

What Does Random Mean?

I’ve been reading a lot of articles about scientific journalism, and what Scientists and Journalists need to change in how they release new findings to combat public misunderstanding. There are some great ideas there, and a lot of people committed to making this happen. The problem, though, is that science has a lot of concepts and vocabulary that are either exclusive to science (and impossible for non-scientists to understand) or are used differently in a colloquial context. So I’m going to start small and pick one. RANDOM.

If you’re having a conversation with someone and the word “random” comes up, you’re likely to think “something completely unexpected,” or “something without precedent,” or “something that just makes no sense.” “Random” started off meaning one thing to scientists and mathematicians and another to everyone else, and now it’s becoming a catchword for many other things that are even further removed from the strictest definition of “random.” Pseudoscientists and peddlers of dubious ideas and products take advantage of this by using the new popular understanding of the word to misrepresent or even mock science that uses it, so I want to set you straight.

Let’s start off with a straightforward explanation. “Random,” in science or mathematics, refers to a set or subset of existing things that is separated, combined, or put in order without any plan or pattern.

Take a look at A Million Random Digits with 100,000 Normal Deviates. This book has been around for a long time, and it’s an important tool for checking probabilities and mathematical formulae to make sure that they work with numbers that have no patterns. It’s not a very exciting read, obviously, but what you will find if you look at it is pages and pages of numbers. In other words, you will not find symbols, color dots, letters, or little drawings of cats. The numbers are random because they cannot be placed in any kind of sequence – as a simple example, you wouldn’t be able to add three to the first digit, six to the next, nine to the next pair, etc.

If you were talking to your friend about this book of numbers that was, like, totally random, your friend might reasonably expect to find those symbols, color dots, letters, or little drawings of cats. But your friend would be wrong; that wouldn’t truly be random since none of those things exist in the set called “numbers.”

So let’s look at this from the point where I see most of the misinterpretation of “random”. . .genetics. I want you to imagine two bags of marbles. I’m not going to specify how many, because we’re not going to get started on the difference between chromosomes and genes or anything like that. We’re just going to be very general and say that each marble represents a piece of genetic information.

two-sacks

One sack is filled with marbles that represent Dad’s genetic information, the other is filled with marbles that represent Mom’s genetic information. Now let’s say that Dad’s marbles are almost all primary colors, but there are a couple of purples, a few greens, one black, and one white. Mom’s marbles are mostly secondary colors, but she does have a smattering of reds, blues, and yellows, and one black and one white. So you reach into the bags blindfolded and grab a handful of each, and this is what you come up with:

two-sets-of-marbles

That’s random (although it’s unlikely that you’re going to get the single black marble and single white marble from each bag. I just wanted to use them.) Do you see any peach pits, or rocks, or silver marbles? Of course not. They weren’t part of the set from which you were randomly selecting. They’re not going to appear out of nowhere – and if they do, it’s not scientifically random.

Now let’s say that we’re going to pair them up. The only rule is going to be that the marble from Dad’s set can’t be paired up with the same color from Mom’s set. In real life, this happens fast, and the number of pairs is significantly bigger, but this is a decent symbolic representation. So across the top are the marbles we got from Dad, and below that are the marbles we got from Mom.

marbles-paired

Randomly we ended up with a unique combination – but still, there is nothing there that wasn’t present in the original set. Randomly we ended up with an extra blue marble from Mom. It could have been an extra orange, purple, green, red, yellow, black, or white marble – but it could never be a peach pit, or a rock, or a silver marble.

You could take all these marbles and put them in different sequences, but nothing is going to change the number of marbles, the colors, or which marbles came from which bag. You might get different pairs of colors, or all the same pairs but in different order.

Randomness, in science or mathematics, means that we have certain things that are givens. A set of numbers will contain nothing but numbers. A set of genes will contain nothing that isn’t already in the genome. Any random thing we look at will be comprised of something very specific that already exists. What makes it random is how it ends up being put together.

I hope that makes sense. Feel free to ask questions or add to the discussion.

Expert Opinions

Expert Opinions

Somewhere in the wilds of the INTARWEBS, I had the audacity to suggest that the opinions of experts actively practicing in a specialized field probably were things we should pay attention to – especially if they were held by a majority of these experts. Well. . .someone whom I would never, ever be so insensitive enough to characterize as dumb as a bag of hammers took great offense at this. His argument, which boiled down to telling me I was a poopy diaper head, was that if several people were strongly invested in an opinion that gave them emotional satisfaction, it should be held in the same high regard as that of a phalanx of well-educated, well-respected researchers whose opinions were informed by well-supported evidence.

Taking this tack would mean that at the next major convention of geologists, the guy who disproved continental drift by taping cut-out paper continents onto a balloon and blowing it up should be sitting at the table for the panel discussion on plate tectonics or continental drift. His opinion is just as valid!

It would mean that the guy who “disproved” that a plane hit the twin towers by hitting a stack of plastic inboxes with the side of his hand was just as credible as a professional engineer with specialized knowledge of airplane technology or the structure and construction of the towers.

Robin Ince posted a much better rebuttal to this idea in his blog entry “The Fascism of Knowing Stuff.” He’s more articulate about it than I could hope to be. He covers a number of reasons why these beliefs are held mostly by people who don’t know stuff, and people who are not afraid of what is known, but how it will be used. Some of his commenters “got it,” as well – it’s much easier to condemn specialized knowledge that you don’t actually have. It’s comforting to see your beliefs confirmed because you don’t understand the much more complex factual information that challenges them.

One expert, one “guru,” isn’t enough to hang all your understanding upon. Believers tend to believe a source and all those who agree with that source regardless of whether or not they themselves have any expertise. But when all or pretty much all the specialists in a field of knowledge say one thing is the most likely explanation, and the only people who challenge them have no background in that field, you really should have some confidence in the experts.

The Heritage Foundation, among other things, is a big anti-global-warming promoter. I sucked it up and watched one of their videos so I could have a cogent argument with a climate change denialist, and the one thing that every single speaker had in common was the admission that he was not a climate scientist. In fact, most of the speakers weren’t scientists at all. One former astronaut claimed that since he had seen the earth from outer space, that was proof enough to him that the earth looked just fine.

The things we know now are far more complex than the things we used to know. They consist of many more specialized pieces. There are few areas in which a general knowledge is sufficient for understanding. One expert can have a different view, or not really be much of an expert at all – but when an opinion is held about a piece of specialized knowledge and is the consensus among the other people who are actively working in that specialized field, it’s a safe bet to take their word over something some guy said on the internet.